Alpha-amylases

ABSTRACT

The present invention relates to alpha-amylases, nucleic acids encoding the alpha-amylases, methods of producing the alpha-amylases, and methods of using the alpha-amylases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/696,979filed Apr. 27, 2015, now U.S. Pat. No. 9,506,047, which is a divisionalof U.S. application Ser. No. 14/064,911 filed Oct. 28, 2013, now U.S.Pat. No. 9,139,822, which is a divisional of U.S. application Ser. No.13/857,431 filed on Apr. 5, 2013, now U.S. Pat. No. 8,591,969, which isa divisional of U.S. application Ser. No. 13/514,727 filed on Jun. 20,2012, now U.S. Pat. No. 8,435,577, which is a 35 U.S.C. 371 nationalapplication of PCT/EP2011/050073 filed Jan. 4, 2011, which claimspriority or the benefit under 35 U.S.C. 119 of European application nos.10150063.5 and 10150062.7 filed Jan. 4, 2010 and Jan. 4, 2010,respectively, and U.S. provisional application Nos. 61/292,324,61/292,327 61/304,092, 61/333,930, 61/354,775, 61/354,817, 61/355,230and 61/362,536 filed Jan. 5, 2010, Jan. 5, 2010, Feb. 12, 2010, May 12,2010, Jun. 15, 2010, Jun. 15, 2010, Jun. 16, 2010 and Jul. 8, 2010,respectively, the contents of which are fully incorporated herein byreference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to alpha-amylases, nucleic acids encodingthe alpha-amylases, methods of producing the alpha-amylases, and methodsof using the alpha-amylases.

BACKGROUND OF THE INVENTION

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1)constitute a group of enzymes, which catalyze the hydrolysis of starchand other linear and branched 1,4-glucosidic oligo- and polysaccharides.

There is a long history of industrial use of alpha-amylases in severalknown applications such as detergent, baking, brewing, starchliquefaction and saccharification, e.g., in the production of highfructose syrups or ethanol. These and other applications utilizealpha-amylases derived from microorganisms, in particular bacterialalpha-amylases.

One of the first bacterial alpha-amylases to be used was analpha-amylase from B. licheniformis, also known as Termamyl™, which hasbeen extensively characterized and the crystal structure has beendetermined for this enzyme. Alkaline amylases, such as the alpha-amylasederived from Bacillus sp. strains NCIB 12289, NCIB 12512, NCIB 12513,and DSM 9375 (disclosed in WO 95/26397), form a particular group ofalpha-amylases that are useful in detergents. Many of these knownbacterial amylases have been modified in order to improve theirfunctionality in a particular application.

Termamyl™ and many highly efficient alpha-amylases require calcium foractivity. The crystal structure of Termamyl™ shows that three calciumatoms are bound to the alpha-amylase structure coordinated by negativelycharged amino acid residues. This requirement for calcium is adisadvantage in applications where strong chelating compounds arepresent, such as in detergents or during ethanol production from wholegrains, where the plant material comprises a large amount of naturalchelators such as phytate.

Calcium-insensitive amylases are known, e.g., the alpha-amylasesdisclosed in EP 1022334 and WO 03/083054, and a Bacillus circulansalpha-amylase having the sequence disclosed in UNIPROT:Q03657.

It would therefore be beneficial to provide alpha-amylases with reducedcalcium sensitivity.

SUMMARY OF THE INVENTION

The present invention relates to alpha-amylases comprising the A- andC-domains of a calcium-sensitive alpha-amylase and the B-domain or apart thereof of a calcium-insensitive alpha-amylase. The alpha-amylaseshave high stability and/or activity in the presence of a strong chelatorand further have considerably improved performance in various industrialapplications.

The invention also relates to compositions comprising the alpha-amylasesof the invention, such as detergent compositions.

In addition, the invention relates to nucleic acids encoding thealpha-amylases of the invention, plasmids comprising such nucleic acids,host cells comprising such a plasmid or nucleic acid, and methods forproducing the alpha-amylases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the alpha-amylases having the amino acidsequences of SEQ ID NOS: 1-16, 29, and 30.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A-, B- and C-Domains: The structure of alpha-amylases comprises threedistinct domains A, B and C, see, e.g., Machius et al., 1995, J. Mol.Biol. 246: 545-559. The term “domain” means a region of a polypeptidethat in itself forms a distinct and independent substructure of thewhole molecule. Alpha-amylases consist of a beta/alpha-8 barrelharboring the active site, which is denoted the A-domain, a rather longloop between the beta-sheet 3 and alpha-helix 3, which is denoted theB-domain, and a C-domain and in some cases also a carbohydrate bindingdomain (e.g., WO 2005/001064; Machius et al., supra).

The domains of an alpha-amylase can be determined by structure analysissuch as by using crystallographically techniques. An alternative methodfor determining the domains of an alpha-amylase is by sequence alignmentof the amino acid sequence of the alpha-amylase with anotheralpha-amylase for which the domains have been determined. The sequencethat aligns with, e.g., the B-domain sequence in the alpha-amylase forwhich the B-domain has been determined can be considered the B-domainfor the given alpha-amylase.

Allelic Variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1)constitute a group of enzymes, which catalyze the hydrolysis of starchand other linear and branched 1,4-glucosidic oligo- and polysaccharides.Alpha-amylases derived from a wide selection of organisms includingbacteria, such as from species of the genus Bacillus, e.g., Bacilluslicheniformis; from species of fungi, such as Aspergillus oryzae(TAKA-amylase) or Aspergillus niger, from plants such as barley and frommammals, are known.

Calcium-insensitive amylase means an alpha-amylase that does not requirethe presence of calcium for optimal activity and/or for maintaining theactive conformation/structure.

Calcium-sensitive amylase means an alpha-amylase that requires thepresence of calcium to retain its structure and/or to have fullenzymatic activity. For some calcium-sensitive amylases it has beenshown that they contain a calcium atom coordinated to acidic amino acidresidues in the active conformation. A large number of calcium-sensitivealpha-amylases are known and have been used industrially because oftheir beneficial properties. Calcium-sensitive alpha-amylases aregenerally sensitive towards conditions that lead to loss of the calciumatom coordinated in their structure such as detergent compositions andfuel mass.

Calcium sensitivity is determined by incubating a given alpha-amylase inthe presence of a strong chelator and analyzing the impact of thisincubation on the activity or stability of the alpha-amylase. Acalcium-sensitive alpha-amylase will be less stable in the presence of achelator or lose a major part or all of its activity by such incubationwhereas a calcium-insensitive alpha-amylase will not lose activity orwill only lose a minor part of the activity during incubation. Chelatorstrength may be evaluated using methods known in the art such as themethods disclosed in Nielsen et al., 2003, Anal. Biochem. 314: 227-234;and Nagarajan and Paine, 1984, J. Am. Oil Chem. Soc. 61(9): 1475-1478,which are incorporated herein by reference. Examples of strong chelatorsthat may be used for such an assay are EGTA (ethylene glycol tetraaceticacid), EDTA (ethylene diamine tetraacetic acid), DTPA (diethylenetriamine pentaacetic acid), DTMPA (diethylene triamine-penta-methylenephosphonic acid) and HEDP (1-hydroxyethan-1,1-diylbis(phosphonic acid)).Other strong chelators may be used to determine the calcium sensitivityof an alpha-amylase. Persons of ordinary skill in the art would be ableto determine the temperature, pH and calcium concentration to use fordetermining calcium sensitivity. Typically, one uses a temperature whichis about 5-10 degrees greater than the temperature optimum.

Coding Sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of its polypeptideproduct. The boundaries of the coding sequence are generally determinedby an open reading frame, which usually begins with the ATG start codonor alternative start codons such as GTG and TTG and ends with a stopcodon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

Control Sequence: The term “control sequence” means all componentsnecessary for the expression of a polynucleotide encoding analpha-amylase of the present invention. Each control sequence may benative or foreign to the polynucleotide encoding the alpha-amylase ornative or foreign to each other. Such control sequences include, but arenot limited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding an alpha-amylase.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression Vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host Cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved Property: The term “improved property” means a characteristicassociated with an alpha-amylase that is improved compared to otheralpha-amylases. Such improved properties include, but are not limitedto, altered temperature-dependent activity profile, thermostability, pHactivity, pH stability, substrate specificity, product specificity, andchemical stability.

Nucleic Acid Construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence.

Operably Linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of the polynucleotide sequence such that thecontrol sequence directs the expression of the coding sequence of apolypeptide.

Parent Enzyme: The term “parent” alpha-amylase means an alpha-amylase towhich modifications are made to produce an alpha-amylase of the presentinvention. The parent may be a naturally occurring (wild-type)polypeptide, or a variant thereof, prepared by any suitable means. Forinstance, the parent protein may be a variant of a naturally occurringpolypeptide which has a modified or altered amino acid sequence. Aparent may also be an allelic variant.

Polypeptide Fragment: The term “polypeptide fragment” means apolypeptide having one or more (several) amino acids deleted from theamino and/or carboxyl terminus of a mature polypeptide; wherein thefragment has alpha-amylase activity. In one aspect, a fragment containsat least 481 amino acid residues, e.g., at least 483, at least 486, andat least 493 amino acid residues.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide sequencehaving one or more (several) nucleotides deleted from the 5′ and/or 3′end of a mature polypeptide coding sequence; wherein the subsequenceencodes a polypeptide fragment having alpha-amylase activity.

Variant: The term “variant” means a polypeptide having alpha-amylaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, of one or more (several) amino acid residues at one ormore (several) positions. A substitution means a replacement of an aminoacid occupying a position with a different amino acid; a deletion meansremoval of an amino acid occupying a position; and an insertion meansadding amino acids, e.g., 1-5 amino acids, adjacent to and following anamino acid occupying a position.

Wild-Type: The term “wild-type” alpha-amylase denotes an alpha-amylaseexpressed by a naturally occurring microorganism, such as a bacterium,yeast or filamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, unless indicated otherwise, thehybrid polypeptide disclosed in SEQ ID NO: 27 (which has the sequence ofamino acids 1-104 of Bacillus stearothermophilus alpha-amylase (SEQ IDNO: 4), followed by amino acids 103-208 of Bacillus circulansalpha-amylase (SEQ ID NO: 13), followed by amino acids 211-515 ofBacillus stearothermophilus alpha-amylase (SEQ ID NO: 4)) is used todetermine the corresponding amino acid residue in another alpha-amylase.The amino acid sequence of another alpha-amylase is aligned with themature polypeptide disclosed in SEQ ID NO: 27, and based on thealignment, the amino acid position number corresponding to any aminoacid residue in the mature polypeptide disclosed in SEQ ID NO: 27 can bedetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofthe EMBOSS package (EMBOSS: The European Molecular Biology Open SoftwareSuite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version3.0.0 or later.

Identification of the corresponding amino acid residue in anotheralpha-amylase can be confirmed by an alignment of multiple polypeptidesequences using “ClustalW” (Larkin et al., 2007, Bioinformatics 23:2947-2948).

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 27 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more (several) representatives in the protein structure databases.Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815;McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilizeinformation from a variety of sources (PSI-BLAST, secondary structureprediction, structural alignment profiles, and solvation potentials) asinput to a neural network that predicts the structural fold for a querysequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol.313: 903-919, can be used to align a sequence of unknown structure withthe superfamily models present in the SCOP database. These alignmentscan in turn be used to generate homology models for the polypeptide, andsuch models can be assessed for accuracy using a variety of toolsdeveloped for that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Eng. 11: 739-747), and implementations of these algorithms canadditionally be utilized to query structure databases with a structureof interest in order to discover possible structural homologs (e.g.,Holm and Park, 2000, Bioinformatics 16: 566-567). These structuralalignments can be used to predict the structurally and functionallycorresponding amino acid residues in proteins within the same structuralsuperfamily. This information, along with information derived fromhomology modeling and profile searches, can be used to predict whichresidues to mutate when moving mutations of interest from one protein toa close or remote homolog.

In describing the alpha-amylase variants of the present invention, thenomenclature described below is adapted for ease of reference. In allcases, the accepted IUPAC single letter or triple letter amino acidabbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine with alanine at position 226 is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, represents asubstitution of glycine (G) with arginine (R) and of serine (S) withphenylalanine (F) at positions 205 and 411, respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:original amino acid, position, original amino acid, new inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. Multiple insertions of aminoacids are designated [Original amino acid, position, original aminoacid, new inserted amino acid #1, new inserted amino acid #2; etc.]. Forexample, the insertion of lysine and alanine after glycine at position195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of tyrosine and glutamic acid for arginine and glycine atpositions 170 and 195, respectively.

Different Alterations.

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginge with tyrosine or glutamic acid atposition 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants:

Tyr167Gly+Arg170Gly, Tyr167Gly+Arg170Ala, Tyr167Ala+Arg170Gly, andTyr167Ala+Arg170Ala.

Alpha-Amylases

The alpha-amylases of the present invention comprise an A-domain of acalcium-sensitive alpha-amylase, a B-domain of a calcium-insensitivealpha-amylase, and a C-domain of a calcium-sensitive alpha-amylase. Theparent alpha-amylase may further comprise a carbohydrate-binding module.

Calcium-Sensitive Alpha-Amylases

Examples of calcium-sensitive alpha-amylases include the followingalpha-amylases:

1. Bacillus amyloliquefaciens alpha-amylase having the amino acidsequence of SEQ ID NO: 1;

2. Bacillus flavothermus amylase, AMY1048 described in WO 2005/001064,having the amino acid sequence of SEQ ID NO: 2;

3. Bacillus licheniformis alpha-amylase having the amino acid sequenceof SEQ ID NO: 3,

4. Bacillus stearothermophilus alpha-amylase having the amino acidsequence of SEQ ID NO: 4;

5. Alpha-amylase AA560 derived from Bacillus sp. DSM 12649 described inWO 00/60060, having the amino acid sequence of SEQ ID NO: 5;

6. Alpha-amylase derived from Bacillus sp. strain NCIB 12512 describedin WO 95/26397, having the amino acid sequence of SEQ ID NO: 6;

7. Alpha-amylase derived from Bacillus sp. strain NCIB 12513 describedin WO 95/26397, having the amino acid sequence of SEQ ID NO: 7;

8. Alpha-amylase SP707 described by Tsukamoto et al., 1988, Biochem.Biophys. Res. Comm. 151: 25-31, having the amino acid sequence of SEQ IDNO: 8;

9. Alpha-amylase TS-22 having the amino acid sequence of SEQ ID NO: 9;

10. Alpha-amylase TS-23 described in J. Appl. Microbiology, 1997, 82:325-334 (SWALL:q59222), having the amino acid sequence of SEQ ID NO: 10;

11. Alpha-amylase derived from Bacillus sp. KSM-AP1378 (FERM BP-3048)described in WO 97/00324, having the amino acid sequence of SEQ ID NO:11;

12. Alpha-amylase derived from Bacillus sp. A 7-7 described in WO02/10356, having the amino acid sequence of SEQ ID NO: 12;

13. Alpha-amylase derived from Bacillus stearothermophilus (SpezymeXtra), having the amino acid sequence of SEQ ID NO: 29.

14. Cytophaga alpha-amylase described in Jeang et al., 2002, Appl.Environ. Microbiol. 68:3651-3654, having the amino acid sequence of SEQID NO: 30; as well as hybrids and variants of any of thesecalcium-sensitive alpha-amylases.

Other calcium-sensitive alpha-amylases include the alpha-amylaseproduced by the B. licheniformis strain described in EP 0252666 (ATCC27811) and the alpha-amylases disclosed in WO 91/00353 and WO 94/18314.

The calcium-sensitive alpha-amylase may be a hybrid of two or morecalcium-sensitive alpha-amylases, such as a hybrid between Bacillusamyloliquefaciens alpha-amylase and Bacillus licheniformisalpha-amylase.

Commercially-available calcium-sensitive alpha-amylases are the productssold under the following tradenames: Optitherm™ and Takatherm™(available from Danisco); Maxamyl™ (available from Danisco), Spezym AA™,Spezyme Delta AA™, Spezyme Fred and Spezyme Xtra (available fromDanisco), and Keistase™ (available from Daiwa), PURASTAR™ ST 5000E, andPURASTAR™ HPAM L (from Genencor Int.).

The A-, B-, C-, and carbohydrate binding domains of thesecalcium-sensitive alpha-amylases are provided in the following table:

C-terminal extension or Carbohy- A-Domain drate (A1 and A2 B- C- BindingAlpha-Amylase Domains) Domain Domain Module Bacillus 1-101 + 208-396102-207 397-483 amyloliquefaciens (SEQ ID NO: 1) Bacillus 1-105 +212-398 106-211 399-484 485-586 flavothermus (SEQ ID NO: 2) Bacillus1-103 + 208-396 104-207 397-483 licheniformis (SEQ ID NO: 3) Bacillus1-104 + 211-396 105-210 397-483 484-515 stearothermophilus (SEQ ID NO:4) Bacillus sp. 1-105 + 213-398 106-212 399-485 (SEQ ID NO: 5) Bacillussp. 1-105 + 213-398 106-212 399-485 NCIB 12512 (SEQ ID NO: 6) Bacillussp. 1-105 + 213-398 106-212 399-485 NCIB 12513 (SEQ ID NO: 7) SP7071-105 + 213-398 106-212 399-485 (SEQ ID NO: 8) TS-22 1-105 + 213-398106-212 399-484 485-586 (SEQ ID NO: 9) TS-23 1-105 + 213-398 106-212399-484 485-583 (SEQ ID NO: 10) Bacillus sp. 1-105 + 213-398 106-212399-485 KSM-AP1378 (SEQ ID NO: 11) Bacillus sp. SP7-7 1-105 + 213-398106-212 399-485 (SEQ ID NO: 12) Bacillus 1-104 + 211-396 105-210 397-483484-486 stearothermophilus alpha-amylase (Spezyme Xtra, SEQ ID NO: 29)Cytophaga 1-102 + 209-397 103-208 398-484 alpha-amylase (SEQ ID NO: 30)Calcium-Insensitive Alpha-Amylases

Examples of calcium-insensitive alpha-amylases include the following:

1. Bacillus circulans alpha-amylase having the sequence shown in SEQ IDNO: 13;

2. KSM K-36 alpha-amylase having the sequence disclosed in SEQ ID NO:14;

3. KSM K-38 alpha-amylase having the sequence disclosed in SEQ ID NO:15;

4. Pyrococcus woesei alpha-amylase having the amino acid sequence of SEQID NO: 16;

5. Pyrococcus hybrid alpha-amylase described in WO 03/083054 having theamino acid sequence of SEQ ID NO: 31;

as well as hybrids and variants of any of these alpha-amylases.

The A-, B-, C-, and carbohydrate binding domains of thesecalcium-insensitive alpha-amylases are provided in the following table:

C-terminal extension or Carbohy- A-Domain drate (A1 and A2 B- C- BindingAlpha-Amylase Domains) Domain Domain Module Bacillus circulans 1-102 +209-395 103-208 396-482 483-492 (SEQ ID NO: 13) KSM K-36 1-103 + 208-393104-207 394-480 (SEQ ID NO: 14) KSM K-38 1-103 + 208-393 104-207 394-480(SEQ ID NO: 15) Pyrococcus woesei 1-109 + 172-338 110-171 339-435 (SEQID NO: 16) Pyrococcus 1-109 + 172-338 110-171 339-435 hybrid alpha-amylase (SEQ ID NO: 31)Alpha-Amylases of the Invention

The alpha-amylases of the present invention comprise an A-domain of acalcium-sensitive alpha-amylase, a B-domain of a calcium-insensitivealpha-amylase, and a C-domain of a calcium-sensitive alpha-amylase.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 1.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 2.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 3.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 4.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 5.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 6.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 7.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 8.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 9.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 10.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 11.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 12.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 29.

In an embodiment, the A-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the A-domain of SEQ ID NO: 30.

In an embodiment, the B-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the B-domain of SEQ ID NO: 13.

In an embodiment, the B-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the B-domain of SEQ ID NO: 14.

In an embodiment, the B-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the B-domain of SEQ ID NO: 15.

In an embodiment, the B-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the B-domain of SEQ ID NO: 16.

In an embodiment, the B-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the B-domain of SEQ ID NO: 31.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 1.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 2.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 3.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 4.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 5.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 6.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 7.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 8.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 9.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 10.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 11.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 12.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 29.

In an embodiment, the C-domain has at least 60% sequence identity, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the C-domain of SEQ ID NO: 30.

The alpha-amylases may be produced by substituting the B-domain or aportion thereof of a calcium-sensitive alpha-amylase with the B-domainor a portion thereof of a calcium-insensitive alpha-amylase. Thealpha-amylases also may be produced by substituting the A- and C-domainsor a portion thereof of a calcium-insensitive alpha-amylase with the A-and C-domains or a portion thereof of a calcium-sensitive alpha-amylase.When producing a hybrid alpha-amylase, no amino acids should be deletedor inserted in the two splicing sites, i.e., the two sites where thesequence of the calcium-sensitive alpha-amylase is combined with thesequence of the calcium-insensitive alpha-amylase.

The boundaries of the A-, B- and C-domains of calcium-sensitive andcalcium-insensitive amylases provided in the tables above are flexible,and some liberty regarding the sequences is permitted. Thus, in generalit is possible to deviate from the exact boundaries for the domains byup to 20 amino acids, e.g., less than 20 amino acids, less than 10 aminoacids, less than 6 amino acids, and less than 3 amino acids. In otherwords, the sequence of the calcium-sensitive alpha-amylase to bereplaced with the sequence of a calcium-insensitive alpha-amylase may bewithin 20 amino acids of the boundaries of the B-domain, e.g., less than10 amino acids, within 6 amino acids, and within 3 amino acids. Forexample, the boundaries differ by one amino acid, two amino acids, threeamino acids, four amino acids, five amino acids, six amino acids, sevenamino acids, eight amino acids, nine amino acids, or ten amino acids.

For example, for the B. amyloliquefaciens alpha-amylase (SEQ ID NO: 1)where the B-domain has been determined as amino acid residues 102-207,the sequence to be replaced by the corresponding sequence of acalcium-insensitive alpha-amylase starts at a position in the range ofpositions 92-112 and ending at a position in the range of positions197-217, e.g., starting at a position in the range of positions 96-108and ending at a position in the range of positions 198-213 or startingat a position in the range of positions 99-105 and ending at a positionin the range of positions 204-210. The A and C-domains of the B.amyloliquefaciens alpha-amylase were determined to be amino acidresidues 1-101 (A1)+208-396 (A2) and 397-483, respectively. Thealpha-amylases of the present invention may comprise an A1-domainstarting at a position in the range of positions 1-5 and ending aposition in the range of positions 91-111, e.g., starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 96-101 or starting at a position in the range of positions 1-3and ending at a position in the range of positions 101-106. Thealpha-amylases of the present invention may comprise A2 and C-domainsstarting at a position in the range of positions 198-218 and ending at aposition in the range of positions 478-483, e.g., starting at a positionin the range of positions 203-208 and ending at a position in the rangeof positions 480-483 or starting at a position in the range of positions208-213 and ending at a position in the range of positions 480-483.

For the B. flavothermus alpha-amylase (SEQ ID NO: 2) where the B-domainhas been determined as amino acid residues 106-211, the sequence to bereplaced by the corresponding sequence of a calcium-insensitivealpha-amylase starts at a position in the range of positions 96-116 andending at a position in the range of positions 198-218, e.g., startingat a position in the range of positions 100-112 and ending at a positionin the range of positions 202-214 or starting at a position in the rangeof positions 103-109 and ending at a position in the range of positions205-212. The A and C-domains of the B. flavothermus alpha-amylase weredetermined to be amino acid residues 1-105 (A1)+212-398 (A2) and399-484, respectively. The alpha-amylases of the present invention maycomprise an A1-domain starting at a position in the range of positions1-5 and ending a position in the range of positions 95-115, e.g.,starting at a position in the range of positions 1-3 and ending at aposition in the range of positions 100-105 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 105-110. The alpha-amylases of the present invention maycomprise A2 and C-domains starting at a position in the range ofpositions 202-222 and ending at a position in the range of positions479-484, e.g., starting at a position in the range of positions 207-212and ending at a position in the range of positions 481-484 or startingat a position in the range of positions 212-217 and ending at a positionin the range of positions 481-484. The B. flavothermus alpha-amylasefurther has a carbohydrate binding domain of amino acid residues485-586. The carbohydrate binding domain is not required for the amylaseactivity and might be fully or partially deleted.

For the B. licheniformis alpha-amylase (SEQ ID NO: 3) where the B-domainhas been determined as amino acid residues 104-207, the sequence of B.licheniformis alpha-amylase to be replaced by the corresponding sequenceof a calcium-insensitive alpha-amylase starts at a position in the rangeof positions 94-114 and ending at a position in the range of positions194-214, e.g., starting at a position in the range of positions 98-110and ending at a position in the range of positions 198-210 or startingat a position in the range of positions 101-107 and ending at a positionin the range of positions 201-207. The A and C-domains of the B.licheniformis alpha-amylase were determined to be amino acid residues1-103 (A1)+208-396 (A2) and 397-483, respectively. The alpha-amylases ofthe present invention may comprise an A1-domain starting at a positionin the range of positions 1-5 and ending a position in the range ofpositions 93-113, e.g., starting at a position in the range of positions1-3 and ending at a position in the range of positions 98-103 orstarting at a position in the range of positions 1-3 and ending at aposition in the range of positions 103-108. The alpha-amylases of thepresent invention may comprise A2 and C-domains starting at a positionin the range of positions 198-218 and ending at a position in the rangeof positions 478-483, e.g., starting at a position in the range ofpositions 203-208 and ending at a position in the range of positions480-483 or starting at a position in the range of positions 208-213 andending at a position in the range of positions 480-483.

For the B. stearothermophilus alpha-amylase (SEQ ID NO: 4) where theB-domain has been determined as amino acid residues 105-210, thesequence to be replaced by the corresponding sequence of acalcium-insensitive alpha-amylase starts at a position in the range ofpositions 95-115 and ending at a position in the range of positions197-213, e.g., starting at a position in the range of positions 99-111and ending at a position in the range of positions 201-213 or startingat a position in the range of positions 102-108 and ending at a positionin the range of positions 204-210. The A and C-domains of the B.stearothermophilus alpha-amylase were determined to be amino acidresidues 1-104 (A1)+211-396 (A2) and 397-483, respectively. Thealpha-amylases of the present invention may comprise an A1-domainstarting at a position in the range of positions 1-5 and ending aposition in the range of positions 94-114, e.g., starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 99-104 or starting at a position in the range of positions 1-3and ending at a position in the range of positions 104-109. Thealpha-amylases of the present invention may comprise A2 and C-domainsstarting at a position in the range of positions 201-221 and ending at aposition in the range of positions 478-483, e.g., starting at a positionin the range of positions 206-211 and ending at a position in the rangeof positions 480-483 or starting at a position in the range of positions211-216 and ending at a position in the range of positions 480-483. TheB. stearothermophilus alpha-amylase further has a C-terminal extensionof amino acid residues 484-586. The C-terminal extension is not requiredfor the amylase activity and might be fully or partially deleted.

For the Bacillus alpha-amylase (SEQ ID NO: 5) where the B-domain hasbeen determined as amino acid residues 106-212, the sequence to bereplaced by the corresponding sequence of a calcium-insensitivealpha-amylase starts at a position in the range of positions 96-116 andending at a position in the range of positions 199-219, e.g., startingat a position in the range of positions 100-112 and ending at a positionin the range of positions 203-215 or starting at a position in the rangeof positions 103-109 and ending at a position in the range of positions206-212. The A and C-domains of the Bacillus alpha-amylase weredetermined to be amino acid residues 1-105 (A1)+213-396 (A2) and399-485, respectively. The alpha-amylases of the present invention maycomprise an A1-domain starting at a position in the range of positions1-5 and ending a position in the range of positions 95-115, e.g.,starting at a position in the range of positions 1-3 and ending at aposition in the range of positions 100-105 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 105-110. The alpha-amylases of the present invention maycomprise A2 and C-domains starting at a position in the range ofpositions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485.

For the Bacillus alpha-amylase (SEQ ID NO: 6) where the B-domain hasbeen determined as amino acid residues 106-212, the sequence to bereplaced by the corresponding sequence of a calcium-insensitivealpha-amylase starts at a position in the range of positions 96-116 andending at a position in the range of positions 199-219, e.g., startingat a position in the range of positions 100-112 and ending at a positionin the range of positions 203-215 or starting at a position in the rangeof positions 103-109 and ending at a position in the range of positions206-212. The A and C-domains of the Bacillus alpha-amylase weredetermined to be amino acid residues 1-105 (A1)+213-398 (A2) and399-485, respectively. The alpha-amylases of the present invention maycomprise an A1-domain starting at a position in the range of positions1-5 and ending a position in the range of positions 95-115, e.g.,starting at a position in the range of positions 1-3 and ending at aposition in the range of positions 100-105 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 105-110. The alpha-amylases of the present invention maycomprise A2 and C-domains starting at a position in the range ofpositions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485.

For the Bacillus alpha-amylase (SEQ ID NO: 7) where the B-domain hasbeen determined as amino acid residues 106-212, the sequence to bereplaced by the corresponding sequence of a calcium-insensitivealpha-amylase starts at a position in the range of positions 96-116 andending at a position in the range of positions 199-219, e.g., startingat a position in the range of positions 100-112 and ending at a positionin the range of positions 203-215 or starting at a position in the rangeof positions 103-109 and ending at a position in the range of positions206-212. The A and C-domains of the Bacillus alpha-amylase weredetermined to be amino acid residues 1-105 (A1)+213-398 (A2) and399-485, respectively. The alpha-amylases of the present invention maycomprise an A1-domain starting at a position in the range of positions1-5 and ending a position in the range of positions 95-115, e.g.,starting at a position in the range of positions 1-3 and ending at aposition in the range of positions 100-105 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 105-110. The alpha-amylases of the present invention maycomprise A2 and C-domains starting at a position in the range ofpositions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485.

For the SP707 alpha-amylase (SEQ ID NO: 8) where the B-domain has beendetermined as amino acid residues 106-212, the sequence to be replacedby the corresponding sequence of a calcium-insensitive alpha-amylasestarts at a position in the range of positions 96-116 and ending at aposition in the range of positions 199-219, e.g., starting at a positionin the range of positions 100-112 and ending at a position in the rangeof positions 203-215 or starting at a position in the range of positions103-109 and ending at a position in the range of positions 206-212. TheA and C-domains of the SP707 alpha-amylase were determined to be aminoacid residues 1-105 (A1)+213-398 (A2) and 399-485, respectively. Thealpha-amylases of the present invention may comprise an A1-domainstarting at a position in the range of positions 1-5 and ending aposition in the range of positions 95-115, e.g., starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 100-105 or starting at a position in the range of positions1-3 and ending at a position in the range of positions 105-110. Thealpha-amylases of the present invention may comprise A2 and C-domainsstarting at a position in the range of positions 203-223 and ending at aposition in the range of positions 482-485, e.g., starting at a positionin the range of positions 208-213 and ending at a position in the rangeof positions 482-485 or starting at a position in the range of positions213-218 and ending at a position in the range of positions 482-485.

For the TS-22 alpha-amylase (SEQ ID NO: 9) where the B-domain has beendetermined as amino acid residues 106-212, the sequence to be replacedby the corresponding sequence of a calcium-insensitive alpha-amylasestarts at a position in the range of positions 96-116 and ending at aposition in the range of positions 199-219, e.g., starting at a positionin the range of positions 100-112 and ending at a position in the rangeof positions 203-215 or starting at a position in the range of positions103-109 and ending at a position in the range of positions 206-212. TheA and C-domains of the TS-22 alpha-amylase were determined to be aminoacid residues 1-105 (A1)+213-398 (A2) and 399-484, respectively. Thealpha-amylases of the present invention may comprise an A1-domainstarting at a position in the range of positions 1-5 and ending aposition in the range of positions 95-115, e.g., starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 100-105 or starting at a position in the range of positions1-3 and ending at a position in the range of positions 105-110. Thealpha-amylases of the present invention may comprise A2 and C-domainsstarting at a position in the range of positions 203-223 and ending at aposition in the range of positions 481-484, e.g., starting at a positionin the range of positions 208-213 and ending at a position in the rangeof positions 482-484 or starting at a position in the range of positions213-218 and ending at a position in the range of positions 482-484. TheTS-22 alpha-amylase further has a carbohydrate binding domain of aminoacid residues 485-586. The carbohydrate binding domain is not requiredfor the amylase activity and might be fully or partially deleted.

For the TS-23 alpha-amylase (SEQ ID NO: 10) where the B-domain has beendetermined as amino acid residues 106-212, the sequence to be replacedby the corresponding sequence of a calcium-insensitive alpha-amylasestarts at a position in the range of positions 96-116 and ending at aposition in the range of positions 199-219, e.g., starting at a positionin the range of positions 100-112 and ending at a position in the rangeof positions 203-215 or starting at a position in the range of positions103-109 and ending at a position in the range of positions 206-212. TheA and C-domains of the TS-23 alpha-amylase were determined to be aminoacid residues 1-105 (A1)+213-398 (A2) and 399-484, respectively. Thealpha-amylases of the present invention may comprise an A1-domainstarting at a position in the range of positions 1-5 and ending aposition in the range of positions 95-115, e.g., starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 100-105 or starting at a position in the range of positions1-3 and ending at a position in the range of positions 105-110. Thealpha-amylases of the present invention may comprise A2 and C-domainsstarting at a position in the range of positions 203-223 and ending at aposition in the range of positions 482-484, e.g., starting at a positionin the range of positions 208-213 and ending at a position in the rangeof positions 482-484 or starting at a position in the range of positions213-218 and ending at a position in the range of positions 482-484. TheTS-23 alpha-amylase further has a carbohydrate binding domain of aminoacid residues 485-583. The carbohydrate binding domain is not requiredfor the amylase activity and might be fully or partially deleted.

For the KSM-AP1378 alpha-amylase (SEQ ID NO: 11) where the B-domain hasbeen determined as amino acid residues 106-212, the sequence to bereplaced by the corresponding sequence of a calcium-insensitivealpha-amylase starts at a position in the range of positions 96-116 andending at a position in the range of positions 199-219, e.g., startingat a position in the range of positions 100-112 and ending at a positionin the range of positions 203-215 or starting at a position in the rangeof positions 103-109 and ending at a position in the range of positions206-212. The A and C-domains of the KSM-AP1378 alpha-amylase weredetermined to be amino acid residues 1-105 (A1)+213-398 (A2) and399-485, respectively. The alpha-amylases of the present invention maycomprise an A1-domain starting at a position in the range of positions1-5 and ending a position in the range of positions 95-115, e.g.,starting at a position in the range of positions 1-3 and ending at aposition in the range of positions 100-105 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 105-110. The alpha-amylases of the present invention maycomprise A2 and C-domains starting at a position in the range ofpositions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485.

For the Bacillus SP7-7 alpha-amylase (SEQ ID NO: 12) where the B-domainhas been determined as amino acid residues 106-212, the sequence to bereplaced by the corresponding sequence of a calcium-insensitivealpha-amylase starts at a position in the range of positions 96-116 andending at a position in the range of positions 199-219, e.g., startingat a position in the range of positions 100-112 and ending at a positionin the range of positions 203-215 or starting at a position in the rangeof positions 103-109 and ending at a position in the range of positions206-212. The A and C-domains of the Bacillus SP7-7 alpha-amylase weredetermined to be amino acid residues 1-105 (A1)+213-398 (A2) and399-485, respectively. The alpha-amylases of the present invention maycomprise an A1-domain starting at a position in the range of positions1-5 and ending a position in the range of positions 95-115, e.g.,starting at a position in the range of positions 1-3 and ending at aposition in the range of positions 100-105 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 105-110. The alpha-amylases of the present invention maycomprise A2 and C-domains starting at a position in the range ofpositions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485.

For the B. stearothermophilus alpha-amylase (SEQ ID NO: 29) where theB-domain has been determined as amino acid residues 105-210, thesequence to be replaced by the corresponding sequence of acalcium-insensitive alpha-amylase starts at a position in the range ofpositions 95-115 and ending at a position in the range of positions197-213, e.g., starting at a position in the range of positions 99-111and ending at a position in the range of positions 201-213 or startingat a position in the range of positions 102-108 and ending at a positionin the range of positions 204-210. The A and C-domains of the B.stearothermophilus alpha-amylase were determined to be amino acidresidues 1-104 (A1)+211-396 (A2) and 397-483, respectively. Thealpha-amylases of the present invention may comprise an A1-domainstarting at a position in the range of positions 1-5 and ending aposition in the range of positions 94-114, e.g., starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 99-104 or starting at a position in the range of positions 1-3and ending at a position in the range of positions 104-109. Thealpha-amylases of the present invention may comprise A2 and C-domainsstarting at a position in the range of positions 201-221 and ending at aposition in the range of positions 478-483, e.g., starting at a positionin the range of positions 206-211 and ending at a position in the rangeof positions 480-483 or starting at a position in the range of positions211-216 and ending at a position in the range of positions 480-483. TheB. stearothermophilus alpha-amylase further has a C-terminal extensionof amino acid residues 484-486. The C-terminal extension is not requiredfor the amylase activity and might be fully or partially deleted.

For the Cytophagus alpha-amylase (SEQ ID NO: 30) where the B-domain hasbeen determined as amino acid residues 103-208, the sequence to bereplaced by the corresponding sequence of a calcium-insensitivealpha-amylase starts at a position in the range of positions 93-113 andending at a position in the range of positions 195-215, e.g., startingat a position in the range of positions 97-109 and ending at a positionin the range of positions 199-211 or starting at a position in the rangeof positions 100-106 and ending at a position in the range of positions202-208. The A and C-domains of the Cytophagus alpha-amylase weredetermined to be amino acid residues 1-102 (A1)+209-397 (A2) and398-484, respectively. The alpha-amylases of the present invention maycomprise an A1-domain starting at a position in the range of positions1-5 and ending a position in the range of positions 92-112, e.g.,starting at a position in the range of positions 1-3 and ending at aposition in the range of positions 97-102 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 102-107. The alpha-amylases of the present invention maycomprise A2 and C-domains starting at a position in the range ofpositions 199-219 and ending at a position in the range of positions479-484, e.g., starting at a position in the range of positions 204-209and ending at a position in the range of positions 481-484 or startingat a position in the range of positions 209-214 and ending at a positionin the range of positions 481-484.

For the Bacillus circulans alpha-amylase (SEQ ID NO: 13), the B-domainhas been determined as amino acid residues 103-208. The alpha-amylasesof the present invention may comprise a B-domain starting at a positionin the range of positions 93-113 and ending at a position in the rangeof positions 195-215, e.g., starting at a position in the range ofpositions 97-109 and ending at a position in the range of positions199-211 or starting at a position in the range of positions 100-106 andending at a position in the range of positions 202-208. The A andC-domains of the Bacillus circulans alpha-amylase were determined to beamino acid residues 1-102 (A1)+209-395 (A2) and 396-482, respectively.The A1-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 1-5 and ending at a position in the range of positions 92-112,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 97-102 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 102-107. The A2-domain which can be replaced by thecorresponding sequence of a calcium-sensitive alpha-amylase starts at aposition in the range of positions 199-219 and ending at a position inthe range of positions 385-405, e.g., starting at a position in therange of positions 204-209 and ending at a position in the range ofpositions 390-395 or starting at a position in the range of positions209-214 and ending at a position in the range of positions 395-400. TheA1 and A2 domains are preferably replaced simultaneously by thecorresponding sequence of a calcium-sensitive alpha-amylase. TheC-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 386-406 and ending at a position in the range of positions477-482, e.g., starting at a position in the range of positions 391-396and ending at a position in the range of positions 479-482 or startingat a position in the range of positions 396-401 and ending at a positionin the range of positions 479-482. The Bacillus circulans alpha-amylasefurther has a C-terminal extension of amino acid residues 483-492. Theextension is not required for the amylase activity and might be fully orpartially deleted.

For the KSM-K36 alpha-amylase (SEQ ID NO: 14), the B-domain has beendetermined as amino acid residues 104-207. The alpha-amylases of thepresent invention may comprise a B-domain starting at a position in therange of positions 93-113 and ending at a position in the range ofpositions 195-215, e.g., starting at a position in the range ofpositions 97-109 and ending at a position in the range of positions199-211 or starting at a position in the range of positions 100-106 andending at a position in the range of positions 202-208. The A andC-domains of the Bacillus circulans alpha-amylase were determined to beamino acid residues 1-103 (A1)+208-393 (A2) and 394-480, respectively.The A1-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 1-5 and ending at a position in the range of positions 93-113,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 98-103 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 103-108. The A2-domain which can be replaced by thecorresponding sequence of a calcium-sensitive alpha-amylase starts at aposition in the range of positions 198-218 and ending at a position inthe range of positions 383-403, e.g., starting at a position in therange of positions 203-208 and ending at a position in the range ofpositions 388-393 or starting at a position in the range of positions208-213 and ending at a position in the range of positions 393-398. TheA1 and A2 domains are preferably replaced simultaneously by thecorresponding sequence of a calcium-sensitive alpha-amylase. TheC-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 384-404 and ending at a position in the range of positions475-480, e.g., starting at a position in the range of positions 389-394and ending at a position in the range of positions 477-480 or startingat a position in the range of positions 394-399 and ending at a positionin the range of positions 477-480.

For the KSM-K38 alpha-amylase (SEQ ID NO: 15), the B-domain has beendetermined as amino acid residues 104-207. The alpha-amylases of thepresent invention may comprise a B-domain starting at a position in therange of positions 93-113 and ending at a position in the range ofpositions 195-215, e.g., starting at a position in the range ofpositions 97-109 and ending at a position in the range of positions199-211 or starting at a position in the range of positions 100-106 andending at a position in the range of positions 202-208. The A andC-domains of the Bacillus circulans alpha-amylase were determined to beamino acid residues 1-103 (A1)+208-393 (A2) and 394-480, respectively.The A1-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 1-5 and ending at a position in the range of positions 93-113,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 98-103 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 103-108. The A2-domain which can be replaced by thecorresponding sequence of a calcium-sensitive alpha-amylase starts at aposition in the range of positions 198-218 and ending at a position inthe range of positions 383-403, e.g., starting at a position in therange of positions 203-208 and ending at a position in the range ofpositions 388-393 or starting at a position in the range of positions208-213 and ending at a position in the range of positions 393-398. TheA1 and A2 domains are preferably replaced simultaneously by thecorresponding sequence of a calcium-sensitive alpha-amylase. TheC-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 384-404 and ending at a position in the range of positions475-480, e.g., starting at a position in the range of positions 389-394and ending at a position in the range of positions 477-480 or startingat a position in the range of positions 394-399 and ending at a positionin the range of positions 477-480.

For the Pyrococcus woesei alpha-amylase (SEQ ID NO: 16), the B-domainhas been determined as amino acid residues 110-171. The alpha-amylasesof the present invention may comprise a B-domain starting at a positionin the range of positions 100-120 and ending at a position in the rangeof positions 161-181, e.g., starting at a position in the range ofpositions 105-115 and ending at a position in the range of positions166-171 or starting at a position in the range of positions 107-113 andending at a position in the range of positions 171-176. The A andC-domains of the Bacillus circulans alpha-amylase were determined to beamino acid residues 1-109 (A1)+172-338 (A2) and 339-435, respectively.The A1-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 1-5 and ending at a position in the range of positions 99-119,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 104-109 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 109-114. The A2-domain which can be replaced by thecorresponding sequence of a calcium-sensitive alpha-amylase starts at aposition in the range of positions 161-181 and ending at a position inthe range of positions 328-348, e.g., starting at a position in therange of positions 167-172 and ending at a position in the range ofpositions 333-338 or starting at a position in the range of positions172-177 and ending at a position in the range of positions 338-343. TheA1 and A2 domains are preferably replaced simultaneously by thecorresponding sequence of a calcium-sensitive alpha-amylase. TheC-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 329-349 and ending at a position in the range of positions430-435, e.g., starting at a position in the range of positions 324-329and ending at a position in the range of positions 432-435 or startingat a position in the range of positions 329-344 and ending at a positionin the range of positions 432-435.

For the Pyrococcus hybrid alpha-amylase (SEQ ID NO: 31), the B-domainhas been determined as amino acid residues 110-171. The alpha-amylasesof the present invention may comprise a B-domain starting at a positionin the range of positions 100-120 and ending at a position in the rangeof positions 161-181, e.g., starting at a position in the range ofpositions 105-115 and ending at a position in the range of positions166-171 or starting at a position in the range of positions 107-113 andending at a position in the range of positions 171-176. The A andC-domains of the Bacillus circulans alpha-amylase were determined to beamino acid residues 1-109 (A1)+172-338 (A2) and 339-435, respectively.The A1-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 1-5 and ending at a position in the range of positions 99-119,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 104-109 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 109-114. The A2-domain which can be replaced by thecorresponding sequence of a calcium-sensitive alpha-amylase starts at aposition in the range of positions 161-181 and ending at a position inthe range of positions 328-348, e.g., starting at a position in therange of positions 167-172 and ending at a position in the range ofpositions 333-338 or starting at a position in the range of positions172-177 and ending at a position in the range of positions 338-343. TheA1 and A2 domains are preferably replaced simultaneously by thecorresponding sequence of a calcium-sensitive alpha-amylase. TheC-domain which can be replaced by the corresponding sequence of acalcium-sensitive alpha-amylase starts at a position in the range ofpositions 329-349 and ending at a position in the range of positions430-435, e.g., starting at a position in the range of positions 334-339and ending at a position in the range of positions 432-435 or startingat a position in the range of positions 339-344 and ending at a positionin the range of positions 432-435.

In an embodiment, the alpha-amylase has a ratio of activity measured bythe Phadebas activity to the activity measured by the G7-pNG assaygreater than 0.1, preferably of more than 0.25, even more preferred morethan 0.5 and most preferred more than 1.

The Phadebas assay is an assay for determining alpha-amylase activityusing a cross-linked insoluble blue-colored starch polymer (Phadebas®Amylase Test, supplied by Magle Life Sciences, Lund, Sweden).

The G7-pNG assay is an assay for determining alpha-amylase activityusing a soluble chromogen compound,p-nitrophenyl-alpha-D-maltoheptaoside. Kits containing PNP-G7 substrateand alpha-Glucosidase is manufactured by Boehringer-Mannheim (cat. no.1054635).

In order to determine the alpha-amylase activity using the Phadebas andthe G7-pNG assays, a reference amylase with known activity must beincluded in the assay and the activity is determined relative to thereference alpha-amylase. For purposes of the present invention, thereference alpha-amylase, which is considered to have the same activitywhen measured by the Phadebas and the G7-pNG assays, is the Bacilluslicheniformis alpha-amylase sold by Novozymes A/S under the tradenameTermamyl®, which has the sequence of SEQ ID NO: 3. Thus, the referencealpha-amylase has a ratio of 1 when measuring the activity by thePhadebas assay relative to the activity measured by the G7-pNP assay.

The ratio of activity on insoluble substrate to activity on solublesubstrate is determined by measuring the activities on the twoparticular selected substrates and calculation of the ratio. Preferablythe ratio is at least 1.5 fold higher than for the parentcalcium-insensitive alpha-amylase, e.g., at least 2 fold higher, atleast 2.5 fold higher and at least 3 fold higher.

Using the methods disclosed below for determining the ratio of activityby the Phadebas assay to the activity of the G7pNG assay, the B.circulans alpha-amylase having the amino acid sequence of SEQ ID NO: 13was found to have a ratio of approximately 0.014.

The inventors have discovered that the calcium sensitivity to asignificant degree can be assigned to the B-domain of ancalcium-sensitive alpha-amylase and that it is possible to retain all orat least some of the beneficial good properties of a calcium-sensitivealpha-amylase by exchanging the complete B-domain or a part of theB-domain of said calcium-sensitive alpha-amylase with the B-domain or apart of the B-domain derived from a calcium-insensitive alpha-amylase.Preferably, the complete B-domain of a calcium-sensitive alpha-amylaseis exchanged with the complete B-domain of a calcium-insensitivealpha-amylase.

The alpha-amylases of the present invention have the benefit of beingless sensitive toward calcium depletion than their parentcalcium-sensitive alpha-amylase but at the same time they maintain theperformance properties of the parent calcium-sensitive alpha-amylase.Calcium sensitivity is manifested in the activity and/or stability ofthe particular alpha-amylase in calcium depleted environments and/orunder acidic conditions. Calcium depleted environments occurs in manyknown applications of alpha-amylases, such as in the presence of strongchelators binding metal ions in particular calcium ions, e.g., indetergents where it is common to include strong chelators because of thebeneficial effect of the laundering process, or in conditions whereplant material including natural chelators such as phytates or citratesis present. Such strong chelators will compete with thecalcium-sensitive alpha-amylases for the calcium ions and will to someextent be able to deprive the calcium-sensitive alpha-amylases for thecalcium ions bound to their structure with the consequence that thestability or activity of the calcium-sensitive is reduced.

Acidic conditions also may affect the stability or activity ofcalcium-sensitive alpha-amylases. It is believed that low pH may lead toa protonation of the amino acid residues that coordinate the calciumions in calcium-sensitive alpha-amylases with the result that they nolonger are capable of binding the calcium and the result is a loss ofstability and/or activity. An example of an application wherealpha-amylases are exposed to acidic conditions is the use ofalpha-amylases in the treatment of digestive disorders such as disclosedin WO 2006/136161 and the use in feed.

Thus, the alpha-amylases have improved stability and/or activity in thepresence of strong chelators and/or improved stability and/or activityat low pH.

The alpha-amylases may further comprise additional substitutions,insertions or deletions known in the art to improve the properties ofalpha-amylases.

For example, oxidizable amino acid residues may be substituted with anon-oxidizable amino acid residue in order to improve the stability ofthe enzyme under oxidizing conditions, e.g., in the presence of bleach,in accordance with the teachings of WO 94/02597 and WO 94/18314, whichare incorporated herein by reference.

In addition, two amino acids in the region 179-182 (using SEQ ID NO: 27numbering) may be deleted to improve stability/activity, as described inWO 96/23873, which is incorporated herein by reference. Two amino acidsat corresponding positions in other alpha-amylases may be deleted.

Further beneficial substitutions that may be introduced are disclosed inWO 99/23211, WO 01/66712 and WO 2006/002643, which are incorporatedherein by reference.

The alpha-amylases of the invention may further comprise additionalsubstitutions, insertions or deletions in the B-domain derived from thecalcium-insensitive alpha-amylase. Examples of suitable substitutions,insertions or deletions in the B-domain of a calcium-insensitivealpha-amylase are the alterations corresponding to the followingalterations in B. circulans alpha-amylase: E179*, N180*, E185W, N186Eand D189T (SEQ ID NO: 13 numbering), which correspond to E181*, N182*,E187W, N188E and D191T in SEQ ID NO: 27 numbering.

In another embodiment, the alpha-amylases of the present inventioncomprise the substitution Q150T.

In another embodiment, the alpha-amylases of the present inventioncomprise the substitution T164V.

In another embodiment, the alpha-amylases of the present inventioncomprise the substitution K184A.

In one embodiment the parent calcium-sensitive alpha-amylase is thealpha-amylase having the amino acid sequence of SEQ ID NO: 7, which hasgood performance in detergents. The B-domain of this calcium-sensitivealpha-amylase may for example be replaced with the B-domain from thecalcium insensitive alpha-amylase of SEQ ID NO: 13. The hybrid mayfurther comprise one or more of the following alterations: E183*, N184*,E189W, N190E, and D193T (SEQ ID NO: 7 numbering), which correspond toE181*, N182*, E187W, N188E, and D191T in SEQ ID NO: 27 numbering. Thesehybrids show good performance in detergents and have improved stabilityin the presence of strong chelators.

In another embodiment the calcium-sensitive alpha-amylase is the B.stearothermophilus alpha-amylase of SEQ ID NO: 4, which has outstandingproperties for liquefaction of starches. The B-domain of thisalpha-amylase may for example be replaced with the B-domain from B.circulans of SEQ ID NO: 13, e.g., the amino acid residues at positions104-209 in SEQ ID NO: 4 may be replaced with the amino acids atpositions 103-208 in SEQ ID NO: 13. The hybrid may optionally compriseone or more of the following modifications: E181*, G182*, E187W, N188E,D191T, S299K, G301R, A302D, D405N, D428N, and P430D (SEQ ID NO: 27numbering).

Other examples of alpha-amylases of the present invention include:

A hybrid where the B-domain of a variant of SEQ ID NO: 5 having thealterations D183*+G184*+R118K+N195F+R320K+R458K (SEQ ID NO: 5 numbering)disclosed in WO 01/66712 is replaced with the B-domain of SEQ ID NO: 13,SEQ ID NO: 14 or SEQ ID NO: 15. For example, amino acids 107-204 of SEQID NO: 14 may replace amino acids 109-208 of said variant of SEQ ID NO:5. In another example, amino acids 104-209 of SEQ ID NO: 15 may replaceamino acids 106-213 of said variant of SEQ ID NO: 5.

A hybrid where part of the B-domain of a variant of SEQ ID NO: 5 havingthe alterations D183*+G184*+R118K+N195F+R320K+R458K (SEQ ID NO: 5numbering) disclosed in WO 01/66712 is replaced with the B-domain of SEQID NO: 14. For example, amino acids 158-204 of SEQ ID NO: 14 may replaceamino acids 160-208 of said variant of SEQ ID NO: 5.

A hybrid where the B-domain of the hybrid mutant alpha-amylase LE399,which has the amino acid sequence of SEQ ID NO: 2 in WO 06/066594 andthe substitutions G46A+T47I+G105A (SEQ ID NO: 3 numbering), is replacedwith the B-domain of SEQ ID NO: 13. For example, amino acids 107-205 ofSEQ ID NO: 13 may replace amino acids 106-202 of LE399.

A hybrid where the B-domain of a variant of SEQ ID NO: 5 having themutations M9L+K118R+G149A+G182T+D183*+G184*+G186A+N195F+M202L+T257I+Y295F+N299Y+R320K+M323T+A339S+E345R+R458K (SEQ ID NO: 5numbering) disclosed in WO 06/002643 is replaced with the B-domain ofSEQ ID NO: 13.

A hybrid where the B-domain of SP707 alpha-amylase of SEQ ID NO: 8 isreplaced with the B-domain of SEQ ID NO: 13.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, a polynucleotide thatis recognized by a host cell for expression of a polynucleotide encodinga polypeptide of the present invention. The promoter sequence containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. colilac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter including a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the host cell of choice may be used in the presentinvention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, whentranscribed is a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′-terminus of the polynucleotide encoding the polypeptide. Anyleader sequence that is functional in the host cell of choice may beused.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. The foreign signal peptide coding sequence may be requiredwhere the coding sequence does not naturally contain a signal peptidecoding sequence. Alternatively, the foreign signal peptide codingsequence may simply replace the natural signal peptide coding sequencein order to enhance secretion of the polypeptide. However, any signalpeptide coding sequence that directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present at theN-terminus of a polypeptide, the propeptide sequence is positioned nextto the N-terminus of a polypeptide and the signal peptide sequence ispositioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the Aspergillus niger glucoamylasepromoter, Aspergillus oryzae TAKA alpha-amylase promoter, andAspergillus oryzae glucoamylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more (several) convenientrestriction sites to allow for insertion or substitution of thepolynucleotide encoding the polypeptide at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the production of a polypeptideof the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negativebacterium. Gram-positive bacteria include, but not limited to, Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.Gram-negative bacteria include, but not limited to, Campylobacter, E.coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207, by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F. A., Passmore, S. M., andDavenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chlysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chlysosporium mops, Chlysosporium keratinophilum, Chlysosporiumlucknowense, Chlysosporium merdarium, Chlysosporium pannicola,Chlysosporium queenslandicum, Chlysosporium tropicum, Chlysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chlysosporium, Phlebia radiata, Pleurotus eiyngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus [Genus]. In a morepreferred aspect, the cell is [Genus species]. In a most preferredaspect, the cell is [Genus species deposit number].

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods well known in the art. Forexample, the cell may be cultivated by shake flask cultivation, andsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing a polypeptide is used as asource of the polypeptide.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotide of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing thepolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or more(several) expression constructs encoding the polypeptide into the planthost genome or chloroplast genome and propagating the resulting modifiedplant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying host cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide may be constitutive or inducible, or may be developmental,stage or tissue specific, and the gene product may be targeted to aspecific tissue or plant part such as seeds or leaves. Regulatorysequences are, for example, described by Tague et al., 1988, PlantPhysiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay inducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a polypeptide. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can alsobe used for transforming monocots, although other transformation methodsare often used for these plants. Presently, the method of choice forgenerating transgenic monocots is particle bombardment (microscopic goldor tungsten particles coated with the transforming DNA) of embryoniccalli or developing embryos (Christou, 1992, Plant J. 2: 275-281;Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,Bio/Technology 10: 667-674). An alternative method for transformation ofmonocots is based on protoplast transformation as described by Omirullehet al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformationmethods for use in accordance with the present disclosure include thosedescribed in U.S. Pat. Nos. 6,395,966 and 7,151,204 (which areincorporated herein by reference).

Following transformation, the transformants comprising the expressionconstruct are selected and regenerated into whole plants according tomethods well known in the art. Often the transformation procedure isdesigned for the selective elimination of selection genes either duringregeneration or in the following generations by using, for example,co-transformation with two separate T-DNA constructs or site specificexcision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the present invention, transgenicplants may be made by crossing a plant having the construct to a secondplant lacking the construct. For example, a construct encoding apolypeptide can be introduced into a particular plant variety bycrossing, without the need for ever directly transforming a plant ofthat given variety. Therefore, the present invention encompasses notonly a plant directly regenerated from cells which have been transformedin accordance with the present invention, but also the progeny of suchplants. As used herein, progeny may refer to the offspring of anygeneration of a parent plant prepared in accordance with the presentinvention. Such progeny may include a DNA construct prepared inaccordance with the present invention, or a portion of a DNA constructprepared in accordance with the present invention. Crossing results inthe introduction of a transgene into a plant line by cross pollinating astarting line with a donor plant line. Non-limiting examples of suchsteps are further articulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Compositions

The present invention also relates to compositions comprising analpha-amylase and at least one additional enzyme. The additionalenzyme(s) may be selected from the group consisting of beta-amylase,cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase),glucoamylase, hemicellulase (e.g., xylanase), isoamylase, isomerase,lipase, phytase, protease, pullulanase, and/or other enzymes useful in acommercial process in conjunction with an alpha-amylase. The additionalenzyme may also be a second alpha-amylase. Such enzymes are known in theart in starch processing, sugar conversion, fermentations for alcoholand other useful end-products, commercial detergents and cleaning aids,stain removal, fabric treatment or desizing, and the like.

Methods of Using the Alpha-Amylases—Industrial Applications

The alpha-amylases of the present invention possess valuable propertiesallowing for a variety of industrial applications. In particular, thealpha-amylases may be used in detergents, in particular laundrydetergent compositions and dishwashing detergent compositions, hardsurface cleaning compositions, and for desizing textiles, fabrics orgarments, production of pulp and paper, beer making, ethanol production,and starch conversion processes.

The alpha-amylases may be used for starch processes, in particularstarch conversion, especially liquefaction of starch (see, e.g., U.S.Pat. No. 3,912,590, EP 063909, EP 252730, WO 96/28567 and WO 99/19467,which are incorporated herein by reference). Also contemplated arecompositions for starch conversion purposes, which may besides thealpha-amylase of the invention also comprise an AMG, pullulanase, andother alpha-amylases.

Further, the alpha-amylases are particularly useful in the production ofsweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017, which isincorporated herein by reference), such as fuel, drinking and industrialethanol, from starch or whole grains.

The alpha-amylases may also be used for desizing of textiles, fabrics,and garments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, and EP119920, which are incorporated herein by reference), beer making orbrewing, and in pulp and paper production or related processes.

Starch Processing

Native starch consists of microscopic granules, which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypical industrial process, the starch has to be thinned or “liquefied”so that it can be suitably processed. This reduction in viscosity isprimarily attained by enzymatic degradation in current commercialpractice.

Conventional starch-conversion processes, such as liquefaction andsaccharification processes are described, e.g., in U.S. Pat. No.3,912,590, EP 252730 and EP 063909, which are incorporated herein byreference.

In an embodiment, the conversion process degrading starch to lowermolecular weight carbohydrate components such as sugars or fat replacersincludes a debranching step.

In the case of converting starch into a sugar, the starch isdepolymerized. Such a depolymerization process consists of, e.g., apre-treatment step and two or three consecutive process steps, i.e., aliquefaction process, a saccharification process, and depending on thedesired end-product, an optional isomerization process.

When the desired final sugar product is, e.g., high fructose syrup thedextrose syrup may be converted into fructose. After thesaccharification process, the pH is increased to a value in the range of6-8, preferably pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immobilized glucose isomerase.

Production of Fermentation Products

In general, alcohol production (ethanol) from whole grain can beseparated into 4 main steps: milling, liquefaction, saccharification,and fermentation.

The grain is milled in order to open up the structure and allow forfurther processing. Two processes used are wet or dry milling. In drymilling, the whole kernel is milled and used in the remaining part ofthe process. Wet milling gives a very good separation of germ and meal(starch granules and protein) and is with a few exceptions applied atlocations where there is a parallel production of syrups.

In the liquefaction process the starch granules are solubilized byhydrolysis to maltodextrins mostly of a DP higher than 4. The hydrolysismay be carried out by acid treatment or enzymatically by analpha-amylase. Acid hydrolysis is used on a limited basis. The rawmaterial can be milled whole grain or a side stream from starchprocessing.

During a typical enzymatic liquefaction, the long-chained starch isdegraded into branched and linear shorter units (maltodextrins) by analpha-amylase. Enzymatic liquefaction is generally carried out as athree-step hot slurry process. The slurry is heated to between 60-95° C.(e.g., 77-86° C., 80-85° C., or 83-85° C.) and the enzyme(s) is (are)added. The liquefaction process is carried out at 85° C. for 1-2 hours.The pH is generally between 5.5 and 6.2. In order to ensure optimalenzyme stability under these conditions, 1 mM of calcium is added (toprovide about 40 ppm free calcium ions). After such treatment, theliquefied starch will have a “dextrose equivalent” (DE) of 10-15.

The slurry is subsequently jet-cooked at between 95-140° C., e.g.,105-125° C., cooled to 60-95° C. and more enzyme(s) is (are) added toobtain the final hydrolysis. The liquefaction process is carried out atpH 4.5-6.5, typically at a pH between 5 and 6. Milled and liquefiedgrain is also known as mash.

Liquefied starch-containing material is saccharified in the presence ofsaccharifying enzymes such as glucoamylases. The saccharificationprocess may last for 12 hours to 120 hours (e.g., 12 to 90 hours, 12 to60 hours and 12 to 48 hours).

However, it is common to perform a pre-saccharification step for about30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to65° C., typically around 60° C. which is followed by a completesaccharification during fermentation referred to as simultaneoussaccharification and fermentation (SSF). The pH is usually between4.2-4.8, e.g., 4.5. In a simultaneous saccharification and fermentation(SSF) process, there is no holding stage for saccharification, rather,the yeast and enzymes are added together.

In a typical saccharification process, maltodextrins produced duringliquefaction are converted into dextrose by adding a glucoamylase and adebranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or apullulanase. The temperature is lowered to 60° C., prior to the additionof a glucoamylase and debranching enzyme. The saccharification processproceeds for 24-72 hours.

Prior to addition of the saccharifying enzymes, the pH is reduced tobelow 4.5, while maintaining a high temperature (above 95° C.), toinactivate the liquefying alpha-amylase. This process reduces theformation of short oligosaccharide called “panose precursors,” whichcannot be hydrolyzed properly by the debranching enzyme. Normally, about0.2-0.5% of the saccharification product is the branched trisaccharidepanose (Glc pα1-6Glc pα1-4Glc), which cannot be degraded by apullulanase. If active amylase from the liquefaction remains presentduring saccharification (i.e., no denaturing), the amount of panose canbe as high as 1-2%, which is highly undesirable since it lowers thesaccharification yield significantly.

Fermentable sugars (e.g., dextrins, monosaccharides, particularlyglucose) are produced by enzymatic saccharification. These fermentablesugars may be further purified and/or converted to useful sugarproducts. In addition, the sugars may be used as a fermentationfeedstock in a microbial fermentation process for producingend-products, such as alcohol (e.g., ethanol and butanol), organic acids(e.g., succinic acid and lactic acid), sugar alcohols (e.g., glycerol),ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g.,lysine), proteins (e.g., antibodies and fragment thereof).

In an embodiment, the fermentable sugars obtained during theliquefaction process steps are used to produce an alcohol, in particularethanol. In ethanol production, an SSF process is commonly used whereinthe saccharifying enzymes and fermenting organisms (e.g., yeast) areadded together and then carried out at a temperature of 30-40° C.

The organism used in fermentation will depend on the desiredend-product. Typically, if ethanol is the desired end product yeast willbe used as the fermenting organism. In some preferred embodiments, theethanol-producing microorganism is a yeast and specificallySaccharomyces such as strains of S. cerevisiae (U.S. Pat. No.4,316,956). A variety of S. cerevisiae are commercially available andinclude but are not limited to FALI (Fleischmann's Yeast), SUPERSTART(Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) and Angelalcohol yeast (Angel Yeast Company, China). The amount of starter yeastemployed in the methods is an amount effective to produce a commerciallysignificant amount of ethanol in a suitable amount of time (e.g., toproduce at least 10% ethanol from a substrate having between 25-40% DSin less than 72 hours). Yeast cells are generally supplied in amounts ofabout 10⁴ to about 10¹², and preferably from about 10⁷ to about 10¹⁰viable yeast count per mL of fermentation broth. After yeast is added tothe mash, it is typically subjected to fermentation for about 24-96hours, e.g., 35-60 hours. The temperature is between about 26-34° C.,typically at about 32° C., and the pH is from pH 3-6, e.g., around pH4-5.

The fermentation may include, in addition to a fermenting microorganisms(e.g., yeast), nutrients, and additional enzymes, including phytases.The use of yeast in fermentation is well known in the art.

In further embodiments, the use of appropriate fermentingmicroorganisms, as is known in the art, can result in a fermentation endproduct such as glycerol, 1,3-propanediol, gluconate,2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid,succinic acid, lactic acid, amino acids, and derivatives thereof. Morespecifically when lactic acid is the desired end product, aLactobacillus sp. (L. casei) may be used; when glycerol or1,3-propanediol is the desired end-product, E. coli may be used; andwhen 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonicacid are the desired end products, Pantoea citrea may be used as thefermenting microorganism. The above enumerated microorganisms are onlyexamples and one skilled in the art will be aware of other fermentingmicroorganisms that may be used to obtain a desired end product.

Processes for Producing Fermentation Products from UngelatinizedStarch-Containing Material

The invention relates to processes for producing fermentation productsfrom starch-containing material without gelatinization (i.e., withoutcooking) of the starch-containing material. The fermentation product,such as ethanol, can be produced without liquefying the aqueous slurrycontaining the starch-containing material and water. In one embodiment aprocess of the invention includes saccharifying (e.g., milled)starch-containing material, e.g., granular starch, below the initialgelatinization temperature, preferably in the presence of alpha-amylaseand/or carbohydrate-source generating enzyme(s) to produce sugars thatcan be fermented into the fermentation product by a suitable fermentingorganism. In this embodiment the desired fermentation product, e.g.,ethanol, is produced from ungelatinized (i.e., uncooked), preferablymilled, cereal grains, such as corn. Accordingly, in the first aspectthe invention relates to processes for producing fermentation productsfrom starch-containing material comprising simultaneously saccharifyingand fermenting starch-containing material using a carbohydrate-sourcegenerating enzyme and a fermenting organism at a temperature below theinitial gelatinization temperature of said starch-containing material.In an embodiment a protease is also present. The protease may be anyacid fungal protease or metalloprotease. The fermentation product, e.g.,ethanol, may optionally be recovered after fermentation, e.g., bydistillation. Typically amylase(s), such as glucoamylase(s) and/or othercarbohydrate-source generating enzymes, and/or alpha-amylase(s), is(are)present during fermentation. Examples of glucoamylases and othercarbohydrate-source generating enzymes include raw starch hydrolyzingglucoamylases. Examples of alpha-amylase(s) include acid alpha-amylasessuch as acid fungal alpha-amylases. Examples of fermenting organismsinclude yeast, e.g., a strain of Saccharomyces cerevisiae. The term“initial gelatinization temperature” means the lowest temperature atwhich starch gelatinization commences. In general, starch heated inwater begins to gelatinize between about 50° C. and 75° C.; the exacttemperature of gelatinization depends on the specific starch and canreadily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material may be determined asthe temperature at which birefringence is lost in 5% of the starchgranules using the method described by Gorinstein and Lii, 1992,Starch/Stärke 44(12): 461-466. Before initiating the process a slurry ofstarch-containing material, such as granular starch, having 10-55 w/w %dry solids (DS), preferably 25-45 w/w % dry solids, more preferably30-40 w/w % dry solids of starch-containing material may be prepared.The slurry may include water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants. Because the process of the invention iscarried out below the initial gelatinization temperature, and thus nosignificant viscosity increase takes place, high levels of stillage maybe used if desired. In an embodiment the aqueous slurry contains fromabout 1 to about 70 vol. %, preferably 15-60 vol. %, especially fromabout 30 to 50 vol. % water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants, or combinations thereof, or the like. Thestarch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids in the starch-containing material are convertedinto a soluble starch hydrolyzate. A process in this aspect of theinvention is conducted at a temperature below the initial gelatinizationtemperature, which means that the temperature typically lies in therange between 30-75° C., preferably between 45-60° C. In a preferredembodiment the process is carried out at a temperature from 25° C. to40° C., such as from 28° C. to 35° C., from 30° C. to 34° C., preferablyaround 32° C. In an embodiment the process is carried out so that thesugar level, such as glucose level, is kept at a low level, such asbelow 6 w/w %, such as below about 3 w/w %, such as below about 2 w/w %,such as below about 1 w/w %., such as below about 0.5 w/w %, or below0.25 w/w %, such as below about 0.1 w/w %. Such low levels of sugar canbe accomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich doses/quantities of enzyme and fermenting organism to use. Theemployed quantities of enzyme and fermenting organism may also beselected to maintain low concentrations of maltose in the fermentationbroth. For instance, the maltose level may be kept below about 0.5 w/w%, such as below about 0.2 w/w %. The process of the invention may becarried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, ormore preferably from pH 4 to 5. In an embodiment fermentation is ongoingfor 6 to 120 hours, in particular 24 to 96 hours.

Processes for Producing Fermentation Products from GelatinizedStarch-Containing Material

In this aspect the invention relates to processes for producingfermentation products, especially ethanol, from a starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.Consequently, the invention relates to processes for producingfermentation products from starch-containing material comprising thesteps of:

(a) liquefying starch-containing material in the presence of analpha-amylase; or

(b) saccharifying the liquefied material obtained in step (a) using acarbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism.

In an aspect, a pullulanase such as a family GH57 pullulanase is alsoused in the liquefaction step. In an embodiment a protease, such as anacid fungal protease or a metalloprotease is added before, during and/orafter liquefaction. In an embodiment the metalloprotease is derived froma strain of Thermoascus, e.g., a strain of Thermoascus aurantiacus,especially Thermoascus aurantiacus CGMCC No. 0670. In an embodiment thecarbohydrate-source generating enzyme is a glucoamylase derived from astrain of Aspergillus, e.g., Aspergillus niger or Aspergillus awamori, astrain of Talaromyces, especially Talaromyces emersonii; or a strain ofAthelia, especially Athelia rolfsii; a strain of Trametes, preferablyTrametes cingulata; a strain of Pachykytospora, e.g., a strain ofPachykytospora papyracea; or a strain of Leucopaxillus, e.g.,Leucopaxillus giganteus; or a strain of Peniophora, e.g., a strain ofthe species Peniophora rufomarginata; or a mixture thereof.Saccharification step (b) and fermentation step (c) may be carried outeither sequentially or simultaneously. A pullulanase and/ormetalloprotease may be added during saccharification and/or fermentationwhen the process is carried out as a sequential saccharification andfermentation process and before or during fermentation when steps (b)and (c) are carried out simultaneously (SSF process). The pullulanaseand/or metalloprotease may also advantageously be added beforeliquefaction (pre-liquefaction treatment), i.e., before or during step(a), and/or after liquefaction (post liquefaction treatment), i.e.,after step (a). The pullulanase is most advantageously added before orduring liquefaction, i.e., before or during step (a). The fermentationproduct, such as especially ethanol, may optionally be recovered afterfermentation, e.g., by distillation. The fermenting organism ispreferably yeast, preferably a strain of Saccharomyces cerevisiae. In aparticular embodiment, the process of the invention further comprises,prior to step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling (e.g., using a hammer mill);

y) forming a slurry comprising the starch-containing material and water.

In a preferred embodiment the particle size is smaller than a #7 screen,e.g., a #6 screen. The aqueous slurry may contain from 10-55 w/w % drysolids (DS), e.g., 25-45 or 30-40 w/w % dry solids (DS) ofstarch-containing material. The slurry is heated to above thegelatinization temperature and an alpha-amylase may be added to initiateliquefaction (thinning). The slurry may be jet-cooked to furthergelatinize the slurry before being subjected to alpha-amylase in step(a). Liquefaction may be carried out as a three-step hot slurry process.The slurry is heated to between 60-95° C., preferably between 70-90° C.,such as preferably between 80-85° C. at pH 4-6, preferably 4.5-5.5, andan alpha-amylase, optionally together with a pullulanase and/orprotease, preferably metalloprotease, are added to initiate liquefaction(thinning). In an embodiment the slurry may then be jet-cooked at atemperature between 95-140° C., preferably 100-135° C., such as 105-125°C., for about 1-15 minutes, preferably for about 3-10 minutes,especially around about 5 minutes. The slurry is cooled to 60-95° C. andmore alpha-amylase and optionally pullulanase and/or protease,preferably metalloprotease, is(are) added to finalize hydrolysis(secondary liquefaction). The liquefaction process is usually carriedout at pH 4-6, in particular at a pH from 4.5 to 5.5. Saccharificationstep (b) may be carried out using conditions well known in the art. Forinstance, a full saccharification process may last up to from about 24to about 72 hours, however, it is common only to do apre-saccharification of typically 40-90 minutes at a temperature between30-65° C., typically about 60° C., followed by complete saccharificationduring fermentation in a simultaneous saccharification and fermentationprocess (SSF process). Saccharification is typically carried out attemperatures from 20-75° C., preferably from 40-70° C., typically around60° C., and at a pH between 4 and 5, normally at about pH 4.5. The mostwidely used process to produce a fermentation product, especiallyethanol, is a simultaneous saccharification and fermentation (SSF)process, in which there is no holding stage for the saccharification,meaning that a fermenting organism, such as yeast, and enzyme(s), may beadded together. SSF may typically be carried out at a temperature from25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to34° C., preferably around about 32° C. In an embodiment fermentation isongoing for 6 to 120 hours, in particular 24 to 96 hours.

Beer Making

The alpha-amylases may also be used in a beer-making process and similarfermentations; the alpha-amylases will typically be added during themashing process. The process is substantially similar to the milling,liquefaction, saccharification, and fermentation processes describedabove.

Starch Slurry Processing with Stillage

Milled starch-containing material is combined with water and recycledthin-stillage resulting in an aqueous slurry. The slurry can comprisebetween 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to40%, 20 to 35% and 30-36% ds). In some embodiments, the recycledthin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to50%, 20 to 40% and also 20 to 30%).

Once the milled starch-containing material is combined with water andbackset, the pH is not adjusted in the slurry. Further the pH is notadjusted after the addition of a phytase and optionally an alpha-amylaseto the slurry. In an embodiment, the pH of the slurry will be in therange of about 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8; 4.5 to5.6; 4.8 to 5.8; 5.0 to 5.8; 5.0 to 5.4; 5.2 to 5.5; and 5.2 to 5.9).The pH of the slurry may be between about 4.5 and 5.2 depending on theamount of thin stillage added to the slurry and the type of materialcomprising the thin stillage. For example, the pH of the thin stillagemay be between 3.8 and 4.5.

During ethanol production, acids can be added to lower the pH in thebeer well, to reduce the risk of microbial contamination prior todistillation.

In some embodiments, a phytase is added to the slurry. In otherembodiments, in addition to a phytase, an alpha-amylase is added to theslurry. In some embodiments, a phytase and alpha-amylase are added tothe slurry sequentially. In other embodiments, a phytase andalpha-amylase are added simultaneously. In some embodiments, the slurrycomprising a phytase and optionally, an alpha-amylase, are incubated(pretreated) for a period of about 5 minutes to about 8 hours (e.g., 5minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15minutes to 4 hours). In other embodiments, the slurry is incubated at atemperature in the range of about 40 to 115° C. (e.g., 45 to 80° C., 50to 70° C., 50 to 75° C., 60 to 110° C., 60 to 95° C., 70 to 110° C., 70to 85° C. and 77 to 86° C.).

In other embodiments, the slurry is incubated at a temperature of about0 to about 30° C. (e.g., 0 to 25° C., 0 to 20° C., 0 to 15° C., 0 to 10°C. and 0 to 5° C.) below the starch gelatinization temperature of thestarch-containing material. In some embodiments, the temperature isbelow about 68° C., below about 65° C., below about 62° C., below about60° C. and below about 55° C. In some embodiments, the temperature isabove about 45° C., above about 50° C., above about 55° C. and aboveabout 60° C. In some embodiments, the incubation of the slurrycomprising a phytase and an alpha-amylase at a temperature below thestarch gelatinization temperature is referred to as a primary (1°)liquefaction.

In one embodiment, the milled starch-containing material is corn ormilo. The slurry comprises 25 to 40% DS, the pH is in the range of 4.8to 5.2, and the slurry is incubated with a phytase and optionally analpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to75° C.

Currently, it is believed that commercially-available microbialalpha-amylases used in the liquefaction process are generally not stableenough to produce liquefied starch substrate from a dry mill processusing whole ground grain at a temperature above about 80° C. at a pHlevel that is less than pH 5.6. The stability of many commerciallyavailable alpha-amylases is reduced at a pH of less than about 4.0.

In a further liquefaction step, the incubated or pretreatedstarch-containing material is exposed to an increase in temperature suchas about 0 to about 45° C. above the starch gelatinization temperatureof the starch-containing material (e.g., 70° C. to 120° C., 70° C. to110° C., and 70° C. to 90° C.) for a period of time of about 2 minutesto about 6 hours (e.g., 2 minutes to 4 hrs, 90 minutes, 140 minutes and90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1hour to 2 hours. The temperature can be increased by a conventional hightemperature jet cooking system for a short period of time, for example,for 1 to 15 minutes. Then the starch may be further hydrolyzed at atemperature in the range of about 75° C. to 95° C. (e.g., 80° C. to 90°C. and 80° C. to 85° C.) for a period of about 15 to 150 minutes (e.g.,30 to 120 minutes). In a preferred embodiment, the pH is not adjustedduring these process steps and the pH of the liquefied mash is in therange of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8; 4.8 to 5.4; and5.0 to 5.2). In some embodiments, a second dose of thermostablealpha-amylase is added to the secondary liquefaction step, but in otherembodiments there is no additional dosage of alpha-amylase.

The incubation and liquefaction steps may be followed bysaccharification and fermentation steps well known in the art.

Distillation

Optionally, following fermentation, an alcohol (e.g., ethanol) may beextracted by, for example, distillation and optionally followed by oneor more process steps.

In some embodiments, the yield of ethanol produced by the methodsprovided herein is at least 8%, at least 10%, at least 12%, at least14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) andat least 23% v/v. The ethanol obtained according to the process providedherein may be used as, for example, fuel ethanol, drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

By-Products

Left over from the fermentation is the grain, which is typically usedfor animal feed either in liquid or dried form. In further embodiments,the end product may include the fermentation co-products such asdistiller's dried grains (DDG) and distiller's dried grain plus solubles(DDGS), which may be used, for example, as an animal feed.

Further details on how to carry out liquefaction, saccharification,fermentation, distillation, and recovery of ethanol are well known tothe skilled person.

According to the process provided herein, the saccharification andfermentation may be carried out simultaneously or separately.

Pulp and Paper Production

The alpha-amylases may also be used in the production of lignocellulosicmaterials, such as pulp, paper and cardboard, from starch reinforcedwaste paper and cardboard, especially where re-pulping occurs at pHabove 7 and where amylases facilitate the disintegration of the wastematerial through degradation of the reinforcing starch. Thealpha-amylases are especially useful in a process for producing apapermaking pulp from starch-coated printed-paper. The process may beperformed as described in WO 95/14807, comprising the following steps:

a) disintegrating the paper to produce a pulp,

b) treating with a starch-degrading enzyme before, during or after stepa), and

c) separating ink particles from the pulp after steps a) and b).

The alpha-amylases may also be very useful in modifying starch whereenzymatically modified starch is used in papermaking together withalkaline fillers such as calcium carbonate, kaolin and clays. With thealpha-amylases it is possible to modify the starch in the presence ofthe filler thus allowing for a simpler integrated process.

Desizing of Textiles, Fabrics and Garments

The alpha-amylases may also be useful in textile, fabric or garmentdesizing. In the textile processing industry, alpha-amylases aretraditionally used as auxiliaries in the desizing process to facilitatethe removal of starch-containing size, which has served as a protectivecoating on weft yarns during weaving. Complete removal of the sizecoating after weaving is important to ensure optimum results in thesubsequent processes, in which the fabric is scoured, bleached and dyed.Enzymatic starch breakdown is preferred because it does not involve anyharmful effect on the fiber material. In order to reduce processing costand increase mill throughput, the desizing process is sometimes combinedwith the scouring and bleaching steps. In such cases, non-enzymaticauxiliaries such as alkali or oxidation agents are typically used tobreak down the starch, because traditional alpha-amylases are not verycompatible with high pH levels and bleaching agents. The non-enzymaticbreakdown of the starch size leads to some fiber damage because of therather aggressive chemicals used. Accordingly, it would be desirable touse the alpha-amylases as they have an improved performance in alkalinesolutions. The alpha-amylases may be used alone or in combination with acellulase when desizing cellulose-containing fabric or textile.

Desizing and bleaching processes are well known in the art. Forinstance, such processes are described in, e.g., WO 95/21247, U.S. Pat.No. 4,643,736, and EP 119920, which are incorporated herein byreference.

Cleaning Processes and Detergent Compositions

The alpha-amylases may be added as a component of a detergentcomposition for various cleaning or washing processes, including laundryand dishwashing. For example, the alpha-amylases may be used in thedetergent compositions described in WO 96/23874 and WO 97/07202.

The alpha-amylases may be incorporated in detergents at conventionallyemployed concentrations. For example, an alpha-amylase of the inventionmay be incorporated in an amount corresponding to 0.00001-10 mg(calculated as pure, active enzyme protein) of alpha-amylase per literof wash/dishwash liquor using conventional dosing levels of detergent.

The detergent composition may for example be formulated as a hand ormachine laundry detergent composition, including a laundry additivecomposition suitable for pretreatment of stained fabrics and a rinseadded fabric softener composition or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

The detergent composition may further comprise one or more otherenzymes, such as a lipase, peroxidase, protease, another amylolyticenzyme, e.g., another alpha-amylase, glucoamylase, maltogenic amylase,CGTase, cellulase, mannanase (such as Mannaway™ from Novozymes,Denmark)), pectinase, pectin lyase, cutinase, and/or laccase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,e.g., a separate additive or a combined additive, can be formulated,e.g., granulate, a liquid, a slurry, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols, fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition may be in any convenient form, e.g., a bar, atablet, a powder, a granule, a paste or a liquid. A liquid detergent maybe aqueous, typically containing up to about 70% water and 0 to about30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of fromabout 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide,ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0 to about 65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleiclacrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system maycomprise peroxy acids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, and thecomposition may be formulated as described in, e.g., WO 92/19708 and WO92/19709.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilre-deposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

The detergent compositions may comprise any enzyme in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.055 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

One or more of the alpha-amylases described herein may additionally beincorporated in the detergent formulations disclosed in WO 97/07202,which is incorporated herein by reference.

This disclosure includes further detail in the following examples, whichare not in any way intended to limit the scope of what is claimed. Thefollowing examples are thus offered to illustrate, but not to limit whatis claimed.

EXAMPLES

Materials and Methods

Fermentation of Alpha-Amylases and Variants

Fermentation may be performed by methods well known in the art or asfollows:

A B. subtilis strain harboring the relevant expression plasmid isstreaked on a LB-agar plate with a relevant antibiotic, and grownovernight at 37° C.

The colonies are transferred to 100 ml BPX media supplemented with arelevant antibiotic (for instance 10 mg/l chloroamphinicol) in a 500 mlshaking flask. Composition of BPX medium:

Potato starch 100 g/l  Barley flour 50 g/l BAN 5000 SKB 0.1 g/l  Sodiumcaseinate 10 g/l Soy Bean Meal 20 g/l Na₂HPO₄, 12 H₂O  9 g/l Pluronic ™0.1 g/l 

BAN is a Bacillus amyloliquefaciens alpha-amylase product sold byNovozymes.

The culture is shaken at 37° C. at 270 rpm for 4 to 5 days.

Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on an UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM acetate pH 5.5. The UF-filtrate is applied onan S-sepharose F.F. and elution is carried out by step elution with 0.2M NaCl in the same buffer. The eluate is dialyzed against 10 mM Tris, pH9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradientfrom 0-0.3 M NaCl over 6 column volumes. The fractions, which containthe activity (measured by the Phadebas assay) are pooled, pH is adjustedto 7.5 and remaining color is removed by a treatment with 0.5% w/volactive coal in 5 minutes.

Phadebas Assay

Alpha-amylase activity is determined by a method employing Phadebas®tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, suppliedby Magle Life Sciences, Lund, Sweden) contain a cross-linked insolubleblue-colored starch polymer, which has been mixed with bovine serumalbumin and a buffer substance and tabletted.

For every single measurement one tablet is suspended in a tubecontaining 5 mL 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, 0.01% TRITON® X100, pHadjusted to the value of interest with NaOH). This is the substratesolution. The alpha-amylase to be tested is diluted in 50 mMBritton-Robinson buffer. This is the amylase solution. The test isperformed at constant temperature, e.g., at room temperature, 37° C. or50° C. The insoluble blue-colored starch polymer is hydrolyzed by thealpha-amylase giving soluble blue fragments. The absorbance of theresulting blue solution, measured spectrophotometrically at 620 nm, is afunction of the alpha-amylase activity.

575 microliters substrate solution is equilibrated at the selectedtemperature for 5 minutes. The hydrolysis is started by adding 25microliters amylase solution to the substrate solution and incubatingthe sample under gentle mixing for 15 minutes at the selectedtemperature. The reaction is stopped by adding 100 microliters 1 M NaOHand immediately cooling on an ice bath after mixing. Aftercentrifugation at 500 g_(av) for 3 minutes, 200 microliters of thesupernatant is transferred to a microtiter plate, and the absorbance at620 nm is read (A_(amyl)). The blind is prepared as described but wherethe 25 microliters amylase solution is replaced by 25 microliters 50 mMBritton-Robison buffer. The absorbance of the blind at 620 nm is A_(b).The standard curve is prepared similarly by making a dilution series ofTermamyl with a known activity and measuring the release of blue colorto the solution as described above. The absorbance of the standards at620 nm is A. The standard curve is a plot of A_(s)-A_(b) against theTermamyl activity in the sample. The activity of the amylase of interestcan be determined by comparing A_(amyl)-A_(b) to the Termamyl standardcurve.

It is important that the measured 620 nm absorbance after 15 minutes ofincubation (testing time) is in the range of 0.2 to 2.0 absorbance unitsat 620 nm. In this absorbance range there is linearity between activityand absorbance (Lambert-Beer law). The dilution of the enzyme (bothamylase of interest and standard) must therefore be adjusted to fit thiscriterion. Under a specified set of conditions (temperature, pH,reaction time, buffer conditions), 1 mg of a given alpha-amylase willhydrolyze a certain amount of substrate and a blue color will beproduced.

G7-pNP Amylase Assay

Alpha-amylase activity may also be determined by a method employing thePNP-G7 substrate. PNP-G7 which is an abbreviation forp-nitrophenyl-alpha-D-maltoheptaoside, is a blocked oligosaccharidewhich can be cleaved by an endo-amylase. Following the cleavage, thealpha-glucosidase included in the kit digests the substrate to liberatea free PNP molecule which has a yellow color and thus can be measured byvisible spectophometry at λ=405 nm (400-420 nm). Kits containing PNP-G7substrate and alpha-glucosidase is manufactured by Roche/Hitachi (cat.no. 11876473). The G7-PNP substrate from this kit contains 22 mmol/L4,6-ethylidene-G7-PNP and 52.4 mmol/L HEPES(2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), pH 7.0) andthe alpha-glucosidase contains 52.4 mmol/L HEPES, 87 mmol/L NaCl, 12.6mmol/L MgCl₂, 0.075 mmol/L CaCl₂, ≧4 kU/L alpha-glucosidase).

The amylase sample to be analyzed is diluted in 50 mM EPPS(4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (Sigma, E9502)),0.01% TRITON® X100, 1 mM CaCl₂, pH 7.0. Before use substrate workingsolution was made by mixing 1 mL of the alpha-glucosidase containingreagent with 0.2 mL 4,6-ethylidene-G7-PNP containing reagent from kit.Immediately after incubation of samples in PCR machine the samples arediluted 10 times in residual activity buffer (50 mM EPPS, 0.01% TRITON®X100, 1 mM CaCl₂, pH7.0). The assay is performed by transferring 20microliters diluted enzyme samples to a 96 well microtiter plate andadding 80 microliters substrate working solution. The solution is mixedand pre-incubated 1 minute at room temperature and absorption ismeasured every 20 sec. over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curveis directly proportional to the specific activity (activity per mgenzyme) of the alpha-amylase in question under the given set ofconditions. The amylase sample should be diluted to a level where theslope is below 0.4 absorbance units per minute.

Enzchek® Amylase Activity Assay

Alpha-amylase activity may also be determined by a method employing theEnzChek® substrate. The substrate in the EnzChek® Ultra Amylase AssayKit (E33651, Invitrogen, La Jolla, Calif., USA) is a corn starchderivative, DQ™ starch, which is corn starch labeled with BODIPY® FL dyeto such a degree that fluorescence is quenched.

One vial containing approx. 1 mg lyophilized substrate is dissolved in100 microliters of 50 mM sodium acetate (pH 4.0). The vial is vortexedfor 20 seconds and left at room temperature, in the dark, withoccasional mixing until dissolved. Then 900 microliters of 100 mMacetate, 0.01% (w/v) TRITON® X100, 0.12 mM CaCl₂, pH 5.5 is added,vortexed thoroughly and stored at room temperature, in the dark untilready to use. The substrate working solution is prepared by diluting10-fold in residual activity buffer (100 mM acetate, 0.01% (w/v) TRITON®X100, 0.12 mM CaCl₂, pH 5.5) giving a substrate concentration of 100micrograms/ml. Immediately after incubation the enzyme is diluted to aconcentration of 20 ng enzyme protein/mL in 100 mM acetate, 0.01% (W/v)TRITON® X100, 0.12 mM CaCl₂, pH 5.5.

For the assay, 25 microliters of the substrate working solution is mixedfor 10 second with 25 microliters of the diluted enzyme in a black 384well microtiter plate. The fluorescence intensity is measured(excitation: 485 nm, emission: 555 nm) once every minute for 15 minutesin each well at 25° C. and the V_(max) is calculated as the slope of theplot of fluorescence intensity against time. The plot should be linearand the residual activity assay has been adjusted so that the dilutedreference enzyme solution is within the linear range of the activityassay.

Example 1: Preparation of Hybrids

The following hybrids of the calcium-sensitive alpha-amylase having thesequence shown in SEQ ID NO: 7 and the calcium-insensitive alpha-amylasehaving the sequence shown in SEQ ID NO: 13 were prepared.

Hybrid 1: the amino acid residues 106-215 of SEQ ID NO: 7 were removedand replaced with the amino acid residues 103-211 of SEQ ID NO: 13,which results in SEQ ID NO: 17, and the following alterations wereintroduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 17numbering), which correspond to E181*, N182*, E187W, N188E and D191Tusing SEQ ID NO: 27 numbering. The sequence of this hybrid is shown inSEQ ID NO: 18.Hybrid 2: the amino acid residues 106-214 of SEQ ID NO: 7 were removedand replaced with the amino acid residues 103-210 of SEQ ID NO: 13,which results in SEQ ID NO: 19, and the following alterations wereintroduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 19numbering), which correspond to E181*, N182*, E187W, N188E and D191Tusing SEQ ID NO: 27 numbering. The sequence of this hybrid is shown inSEQ ID NO: 20.Hybrid 3: the amino acid residues 106-213 of SEQ ID NO: 7 were removedand replaced with the amino acid residues 103-209 of SEQ ID NO: 13,which results in SEQ ID NO: 21, and the following alterations wereintroduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 21numbering), which correspond to E181*, N182*, E187W, N188E and D191Tusing SEQ ID NO: 27 numbering. The sequence of this hybrid is shown inSEQ ID NO: 22.

Example 2: Stability in the Presence of Chelator

Enzyme samples were incubated in buffer pH 8.0 (50 mM EPPS, 0.01%TRITON® X100, pH 8.0) with 1.5% final concentration of DTPA at 49° C.for 1 hour and reference samples were incubated at 4° C. for 1 hour. Inaddition, enzyme samples were incubated in buffer pH 10.0 (50 mM EPPS,0.01% TRITON® X100, pH 10.0) with 1.5% final concentration of DTPA at42° C. for 1 hour and reference samples were incubated at 4° C. for 1hour.

For the determination of amylase stability in buffer pH 8 and pH 10 withDTPA the enzymes to be tested were adjusted to 0.25 and 0.5 mg enzymeprotein/mL by diluting in 5 mM EPPS, 0.01% TRITON® X100, pH 8.0.

160 microliters stability buffer (50 mM EPPS, 0.01% TRITON® X100, 1.875%DTPA, pH 8.0 or pH 10.0) and 40 microliters of the amylase solution weretransferred to a 96-well PCR microtiter plate in duplicate and thecontent was mixed for 1 minute. Final concentration of DTPA was 1.5% ineach well. 20 microliters from each well was transferred to a new PCRmicrotiter plate, which was placed at 4° C. (reference sample). The PCRMTP was incubated in PCR machine for 1 hour at 49° C. when buffer had pH8.0 (pH 8, 49° C. samples) and for 1 hour at 42° C. when buffer had pH10.0 (pH 10, 42° C. samples).

Immediately after incubation, the samples on PCR plates were analyzedfor amylase activity as described in the G7-pNP Amylase assay. It shouldbe noted that in order to reduce interference from DTPA on the assay,both reference and pH 8, 49° C. samples/pH 10, 42° C. samples werediluted to the same concentration before being analyzed for residualactivity. The activity of both the reference samples and the pH 8, 49°C. samples or pH 10, 42° C. samples were determined on the same 96 wellplate. The residual activity was calculated as 100*V_(max) (pH 8, 42° C.or pH 10, 49° C. sample)/V_(max)(reference sample).

Residual activity Residual activity in % after 1 in % after 1 hour 49°C., hour 42° C., pH 8.0 and 1.5% pH 10.0 and 1.5% Enzyme DTPA DTPA SEQID NO: 7 with the 20 18 deletions D183* + T184* (SEQ ID NO: 7 numbering)SEQ ID NO: 7 1 9 SP707 (SEQ ID NO: 8) 1 3 Hybrid 1 100 102 Hybrid 2 102102 Hybrid 3 103 103

Hybrids 1, 2 and 3 are highly stable and have 100% residual activityafter incubation for 1 hour at both pH 8, 49° C. and pH 10, 42° C. Incomparison SEQ ID NO: 7 with the deletions D183*+T184*has less than 20%residual activity at these conditions and SEQ ID NO: 7 and SP707 haveeven less residual activity.

Example 3: Additional Alpha-Amylases

The following alpha-amylases were prepared:

Hybrid 4: the amino acid residues 106-212 of SEQ ID NO: 5 were removedand replaced with the amino acid residues 103-208 of SEQ ID NO: 13,which results in SEQ ID NO: 23, and the following alterations wereintroduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 23numbering), which correspond to E181*, N182*, E187W, N188E and D191T inSEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ IDNO: 24.Hybrid 5: the amino acid residues 106-212 of SEQ ID NO: 8 were removedand replaced with the amino acid residues 103-208 of SEQ ID NO: 13,which results in SEQ ID NO: 25, and the following alterations wereintroduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 25numbering), which correspond to E181*, N182*, E187W, N188E and D191T inSEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ IDNO: 26.

Hybrids 4 and 5 (SEQ ID NOS: 24 and 26), a variant of SEQ ID NO: 5 withthe alterations E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 5numbering), which correspond to E181*, N182*, E187W, N188E and D191T inSEQ ID NO: 27 numbering, and the alpha-amylase of SEQ ID NO: 8 wereincubated with DTPA as described in Example 2. The results show thathybrids 4 and 5 had almost 100% residual activity after the incubations,whereas the other alpha-amylases lost most of their activity during theincubations.

Example 4: Stability of Alpha-Amylase Variants

The stability of a reference alpha-amylase with the amino acid sequenceof SEQ ID NO: 28 (a hybrid of Bacillus stearothermophilus and Bacilluscirculans alpha-amylases (SEQ ID NO: 27) with the alterationsE181*+G182*+E187W+N188E+D191T+D407N+D430N+P432D) and alpha-amylasevariants thereof was determined by incubating the referencealpha-amylase and variants at pH 4.5 and 5.5 and temperatures of 75° C.and 85° C. with 0.12 mM CaCl₂ followed by residual activitydetermination using the EnzChek® substrate (EnzChek® Ultra Amylase assaykit, E33651, Molecular Probes, Invitrogen, La Jolla, Calif., USA).

Purified enzyme samples were diluted to working concentrations of 0.5and 1 or 5 and 10 ppm (micrograms/ml) in enzyme dilution buffer (10 mMacetate, 0.01% TRITON® X100, 0.12 mM CaCl₂, pH 5.0). Twenty microlitersenzyme sample was transferred to 48-well PCR MTP and 180 microlitersstability buffer (150 mM acetate, 150 mM MES, 0.01% TRITON® X100, 0.12mM CaCl₂, pH 4.5 or 5.5) was added to each well and mixed. The assay wasperformed using two concentrations of enzyme in duplicates. Beforeincubation at 75° C. or 85° C., 40 microliters was withdrawn and storedon ice as reference samples. Incubation was performed in a PCR machinefor 30/45 minutes (pH 4.5 and 75° C.), 45/60 minutes (pH 5.5 and 75°C.), 5/10 minutes (pH 4.5 and 85° C.) and 10 minutes (pH 5.5 and 85°C.).

After incubation, the reference samples and samples from the PCR machinewere diluted to 20 ng/ml in residual activity buffer (100 mM acetate,0.01% TRITON® X100, 0.12 mM CaCl₂, pH 5.5) and 25 microliters dilutedenzyme was transferred to black 384-MTP. Residual activity wasdetermined using the EnzChek® substrate as described in the section forthe Enzchek® amylase activity assay. In brief, 25 microliters substrateworking solution (100 micrograms/ml) is added to each well with dilutedenzyme. Fluorescence was determined every minute for 15 minutes usingexcitation filter at 485-P nm and emission filter at 555 nm(fluorescence reader is Polarstar, BMG). The residual activity wasnormalized to control samples for each setup.

Assuming logarithmic decay the half life time (T½ (min)) was calculatedusing the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T is theassay incubation time in minutes, and % RA is the % residual activitydetermined in the assay.

Using this assay setup the half life time was determined for thereference alpha-amylase and variants thereof as shown in Table 1.

TABLE 1 T½ T½ T½ T½ (min) (min) (min) (min) (pH 4.5, (pH 5.5, (pH 4.5,(pH 5.5, 75° C., 75° C., 85° C., 85° C., 0.12 0.12 0.12 0.12 Mutations(SEQ ID NO: mM mM mM mM 27 numbering) CaCl₂) CaCl₂) CaCl₂) CaCl₂)Reference Alpha-Amylase 14 ± 2 33 ± 4 1.8 ± 0.2 4.5 ± 0.2 ReferenceAlpha-Amylase 49 4.2 13 with the substitutions M8L + N105D + K184AReference Alpha-Amylase 58 4.7 15 with the substitutions A27Q + Q86S +A90S + N105D + K184A Reference Alpha-Amylase 59 6.8 28 with thesubstitutions S34K + N105D + K184A + S242Q Reference Alpha-Amylase 505.2 15 with the substitutions R52G + S53Y + N105D + K184A ReferenceAlpha-Amylase 91 11.3 29 with the substitutions V59A + A100G + N105D +T164V + K184A + M307L Reference Alpha-Amylase >120 14.1 39 with thesubstitutions V59A + N105D + Q150T + T164V + K184A + S242Q + M307LReference Alpha-Amylase 108 9.4 24 with the substitutions T80D + N105D +T164V + K184A + M307L Reference Alpha-Amylase 51 6.3 19 with thesubstitutions A91L + N105D + K184A Reference Alpha-Amylase 69 7.3 25with the substitutions A100L + N105D + T164V + K184A + Y222V + M307LReference Alpha-Amylase 32 129 4 <15 with the substitution N105DReference Alpha-Amylase 45 5.5 21 with the substitutions N105D + K117D +Q150T + K184A + S301K + G303R + A304D Reference Alpha-Amylase 20 3 6with the substitutions N105D + E129V + R177L + A179E ReferenceAlpha-Amylase 64 5 19 with the substitutions N105D + E132D + K184AReference Alpha-Amylase 33 3.3 9 with the substitutions N105D + F134E +K184A Reference Alpha-Amylase 44 3.9 16 with the substitutions N105D +E135N + A179N + K184A Reference Alpha-Amylase 27 117 4 10 with thesubstitutions N105D + Q150T Reference Alpha-Amylase 59 7.3 20 with thesubstitutions N105D + Q150T + T164V + F166W + A168E + E171K + K184A +N407D + N430D + D432P Reference Alpha-Amylase >120 13.6 >40 with thesubstitutions N105D + Q150T + T164V + K184A + S242Q + M284T + M307LReference Alpha-Amylase 114 13.4 >40 with the substitutions N105D +Q150T + T164V + K184A + S242Q + M284T + N407D Reference Alpha-Amylase 37141 5 13 with the substitutions N105D + Q150T + F166W + A168E + E171KReference Alpha-Amylase 68 7.9 21 with the substitutions N105D + Q150T +F166W + A168E + E171K + K184A Reference Alpha-Amylase 46 207 5.1 15 withthe substitutions N105D + Q150T + K184A Reference Alpha-Amylase 42 4.714 with the substitutions N105D + Q150T + K184A + Y206M ReferenceAlpha-Amylase 41 111 4.6 16 with the substitutions N105D + Q150T +K184A + S301K + G303R + A304D Reference Alpha-Amylase 50 5.2 15 with thesubstitutions N105D + N157Y + E159Y + H160Y + K184A + H208Y + D210YReference Alpha-Amylase 38 4.3 13 with the substitutions N105D + H160Y +K184A Reference Alpha-Amylase 71 7.5 18 with the substitutions N105D +T164V + K184A + Y222V + M307L Reference Alpha-Amylase >120 11.7 39 withthe substitutions N105D + T164V + K184A + S242Q + M284T + M307LReference Alpha-Amylase 68 5.7 19 with the substitutions N105D + T164V +K184A + F244Y + M284T + M307L Reference Alpha-Amylase 105 12 >40 withthe substitutions N105D + T164V + K184A + M284Q + M307L ReferenceAlpha-Amylase >120 14.6 >40 with the substitutions N105D + T164V +K184A + M284V + M307L Reference Alpha-Amylase 70 230 8.7 23 with thesubstitutions N105D + T164V + K184A + M307L Reference Alpha-Amylase 49140 4 13 with the substitutions N105D + F166W + A168E + E171K ReferenceAlpha-Amylase 33 112 4.8 14 with the substitutions N105D + F166W +A168E + E171K + S301K + G303R + A304D Reference Alpha-Amylase 68 5.8 19with the substitutions N105D + A179D + K184A Reference Alpha-Amylase 565.3 >40 with the substitutions N105D + A179N + K184A ReferenceAlpha-Amylase 56 6 28 with the substitutions N105D + A179Q + K184AReference Alpha-Amylase 40 153 5.6 14 with the substitutions N105D +K184A Reference Alpha-Amylase 55 5.8 15 with the substitutions N105D +K184A + D210V Reference Alpha-Amylase 14 37 1.4 4 with the substitutionsN105D + K184A + A235T Reference Alpha-Amylase 24 83 2.9 9 with thesubstitutions N105D + K184A + S242E Reference Alpha-Amylase 60 183 7 19with the substitutions N105D + K184A + S242Q Reference Alpha-Amylase 9110.8 30 with the substitutions N105D + K184A + S242Q + A235W ReferenceAlpha-Amylase 91 11 30 with the substitutions N105D + K184A + S242Q +G282W Reference Alpha-Amylase 64 5.2 16 with the substitutions N105D +K184A + P245A Reference Alpha-Amylase 46 5.5 16 with the substitutionsN105D + K184A + P245K Reference Alpha-Amylase 38 110 4.7 15 with thesubstitutions N105D + K184A + S301K + G303R + A304D ReferenceAlpha-Amylase 46 5.5 16 with the substitutions N105D + K184A + P348TReference Alpha-Amylase 47 5.2 18 with the substitutions N105D + K184A +P386E Reference Alpha-Amylase 46 5.4 18 with the substitutions N105D +K184A + P386Q Reference Alpha-Amylase 49 5.5 17 with the substitutionsN105D + K184A + P386T Reference Alpha-Amylase 44 3.9 16 with thesubstitutions N105D + K184A + P386V Reference Alpha-Amylase 52 5.7 17with the substitutions N105D + K184A + L388I Reference Alpha-Amylase 645.2 16 with the substitutions N105D + K184A + L388V ReferenceAlpha-Amylase 45 5.6 17 with the substitutions N105D + K184A + N407D +N430D + D432P Reference Alpha-Amylase 52 5.5 17 with the substitutionsN105D + K184A + D432P Reference Alpha-Amylase 47 5.2 18 with thesubstitutions N105D + K184A + T459P Reference Alpha-Amylase 8 27 4 withthe substitutions N105D + Y206K Reference Alpha-Amylase 18 81 2.2 11with the substitutions N105D + Y206M Reference Alpha-Amylase 37 4.1 11with the substitutions N105D + K220P + N224L Reference Alpha-Amylase 23116 2.9 10 with the substitutions N105D + S301K + G303R + A304DReference Alpha-Amylase 23 2 6 with the substitutions V115W + F134Y +E135Q + K169S + G170R + R172L + G174R + F176Y Reference Alpha-Amylase 1737 2 5 with the substitution K117D Reference Alpha-Amylase 9 21 with thesubstitutions E129V + Q150T Reference Alpha-Amylase 21 78 2.2 7 with thesubstitutions F134Y + E135Q + K169S + G170R + R172L + G174R + F176YReference Alpha-Amylase 32 90 4 10 with the substitution Q150T ReferenceAlpha-Amylase 21 49 3 7 with the substitution T164V ReferenceAlpha-Amylase 30 52 3.7 <15 with the substitution K184A ReferenceAlpha-Amylase with the substitutions K184A + I204L ReferenceAlpha-Amylase 8 with the substitutions K184A + I270L ReferenceAlpha-Amylase 23 74 3 9 with the substitution Y206M ReferenceAlpha-Amylase 8 18 3 with the substitution S242E Reference Alpha-Amylase8 18 3 with the substitutions S301K + G303R + A304D ReferenceAlpha-Amylase 8 20 3 with the substitution G475K Reference Alpha-Amylase15 with the substitution G475Q

The results demonstrate that the alpha-amylase variants have asignificantly greater half-life and stability than the referencealpha-amylase.

Example 5: Production of Ethanol Using Alpha-Amylase Variants

Three small scale mashes of a Bacillus stearothermophilus alpha-amylasevariant sold by Novozymes under the name LIQUOZYME SC® and twoalpha-amylase variants described in Example 4 were prepared as follows:about 54 g corn ground, about 51 g tap water, and about 45 g backsetwere mixed in a 250 mL plastic bottle to a total slurry weight of 150 g.The pH of the corn slurry was adjusted to 4.5. The enzymes were added tothe mashes at 2 micrograms of amylase per gram of dry solids. Forliquefaction, the alpha-amylases were added to the bottles and thebottles were mixed thoroughly and placed into a preheated 85° C. waterbath. The samples were held in the water bath for 2 hours at pH 4.5while being shaken every 10 minutes for the first 30 minutes and every30 minutes thereafter for the remainder of the 2 hour liquefaction. Thesamples were then cooled in an ice bath; pH was adjusted to 5.0, and0.75 mL urea and 0.45 mL penicillin were added to reach finalconcentrations of 1000 and 3 ppm in the mashes, respectively. Thesamples were then subjected to simultaneous saccharification andfermentation (SSF) with Spirizyme Fuel® (a glucoamylase product sold byNovozymes).

Five gram aliquots of the mashes were transferred into pre-weighedconical centrifuge tubes, using 5 replicates per mash. SSF was thenperformed on these mashes in a 32° C. water bath for 54 hours usingSpirizyme Fuel®. The glucoamylase dose was 0.50 AGU/g DS for allfermentations. The CO₂ weight loss during SSF was measured and ethanolwas quantified using HPLC after 54 hours of SSF. The average 54 hourHPLC SSF data are provided in Table 2 below.

TABLE 2 Ethanol Yields After 54 Hours Fermentation Alpha-AmylaseEthanol, g/L Std dev. LIQUOZYME SC ® 105.5946 0.3708 ReferenceAlpha-Amylase disclosed in Example 4 115.6339 0.5562 with thesubstitution N105D Reference Alpha-Amylase disclosed in Example 4116.7224 0.8226 with the substitution K184A

The results demonstrate that the use of the alpha-amylase variantsresulted in a significantly greater yield of ethanol relative toLIQUOZYME SC®.

Example 6: Wash Performance in a Detergent

In order to assess the wash performance of alpha-amylases in adetergent, washing experiments were performed. The performance ofhybrids 1, 2 and 3 of Example 1 was tested using the Mini Wash Assay andcompared to the alpha-amylase having the amino acid sequence of SEQ IDNO: 7 with the deletions D183*+T184*(SEQ ID NO: 7 numbering). In thistest, the wash performance of enzyme-detergent solutions can be examinedat several enzyme dosages simultaneously.

Description of the Mini Wash Assay

The Mini Wash has a number of beakers with each beaker able to hold upto 80 ml enzyme-detergent solution. Water hardness is adjusted to 10° dHby addition of CaCl₂, MgCl₂, and NaHCO₃ to the test system. A textilesample, in this case CS-28, is attached to a frame designed to dip thetextile into the enzyme-detergent solution with a frequency of 40submersions per min. The temperature of the enzyme-detergent solution iscontrolled during wash. After wash the textile is rinsed in running tapwater and subsequently dried in the dark. The wash performance of theenzyme-detergent solution is evaluated by measuring the remission at 460nm with a ZEISS MCS 521 VIS Spectrophotometer.

Textiles:

CS-28 is a technical rice starch stained cotton textile that can beobtained from Center For Test materials By, P.O. Box 120, 3133 KTVlaardingen, the Netherlands.

The experiment was conducted under the experimental conditions specifiedbelow:

Detergent Commercial Tide 2X Ultra, inactivated by boiling for 15minutes Detergent dosage 0.78 g/L Test solution volume 60 mL pH Afterwash pH was measured to 8.3 Wash time 20 minutes followed by 5 minutesrinse Temperature 40° C. Water hardness 10° dH, Ca/Mg 3:1 Enzymeconcentration 0; 0.03; 0.06; 0.12; 0.20; 1.0 mg purified in testsolution enzyme protein/L Enzymes SEQ ID NO: 7 with the deletionsD183* + T184* Hybrid 1 Hybrid 2 Hybrid 3 Test material CS-28 (Ricestarch on cotton)Results and Discussion:

The wash performance of the alpha-amylases was normalized to the washperformance of the alpha-amylase having the amino acid sequence of SEQID NO: 7 with the deletions D183*+T184*.

SEQ ID NO: 7 with the Dose (mg enzyme deletions protein/L) D183* + T184*Hybrid 1 Hybrid 2 Hybrid 3 0 100.0 100.0 100.0 100.0 0.03 100.0 108.8105.6 93.7 0.06 100.0 109.4 103.4 92.9 0.12 100.0 108.7 104.5 90.4 0.2100.0 106.2 99.6 94.4 1 100.0 101.2 99.2 96.7

Example 7: Preparation of Hybrids

The following hybrids were prepared.

Hybrid 6: the amino acid residues 106-213 in a variant of SEQ ID NO: 5having the substitutionsM9L+R118K+G149A+G182T+G186A+D183*+G184*+N195F+M202L+V214V+T257I+Y295F+N299Y+R320K+M323T+A339S+E345R+R458K(using SEQ ID NO: 5 numbering) were removed and replaced with the aminoacid residues 103-209 of SEQ ID NO: 13, and the following alterationswere introduced: E182*, N183*, E188W, N189E and D192T, which correspondto E181*, N182*, E187W, N188E and D191T using SEQ ID NO: 27 numbering.The sequence of this hybrid is shown in SEQ ID NO: 32.Hybrid 7: the amino acid residues 106-213 in a variant of SEQ ID NO: 8(using SEQ ID NO: 8 numbering) were removed and replaced with the aminoacid residues 103-209 of SEQ ID NO: 13. The sequence of this hybrid isshown in SEQ ID NO: 33.Hybrid 8: the amino acid residues 104-208 in a variant of SEQ ID NO: 3having amino acids 1-35 replaced by amino acids 1-33 of SEQ ID NO: 1 andhaving the substitutionsG48A+T49I+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S (using SEQ ID NO: 3numbering) were removed and replaced with the amino acid residues103-209 of SEQ ID NO: 13, and the following alterations were introduced:N102D, Q147T, E178*, N179*, K181A, E184W, N185E and D188T, whichcorrespond to N105D, Q150T, E181*, N182*, K184A, E187W, N188E and D191Tusing SEQ ID NO: 27 numbering. The sequence of this hybrid is shown inSEQ ID NO: 34.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention is further defined in the following paragraphs:

Paragraph 1. An isolated alpha-amylase comprising an amino acid sequencehaving at least 80% sequence identity to the B-domain of a parentcalcium-insensitive alpha-amylase, further having a higher ratio ofactivity measured by the Phadebas assay to the activity measured by theG7-pNG assay of more than 0.1, preferably of more than 0.2, even morepreferred more than 0.5, and most preferred more than 1.Paragraph 2. The alpha-amylase of paragraph 1, with at least 80%sequence identity to the sequence of amino acids 105-210 of SEQ ID NO:13.Paragraph 3. An isolated alpha-amylase comprising the A- and C-domainsof a calcium-sensitive alpha-amylase and the B-domain of acalcium-insensitive alpha-amylase.Paragraph 4. The alpha-amylase of any of the preceding paragraphs,further comprising one or more substitutions, insertions or deletions.Paragraph 5. The alpha-amylase of paragraph 3, wherein thecalcium-sensitive alpha-amylase is SEQ ID NO: 7 and thecalcium-insensitive alpha-amylase is SEQ ID NO: 13, and thealpha-amylase optionally further comprises one or more of the followingalterations: D183*+G184*, E189W, N190E, D193T, E189W+N190E+D193T, L217E,Y208M, R119D and W189E+L217E.Paragraph 6. The alpha-amylase of paragraph 3, wherein thecalcium-sensitive alpha-amylase is SEQ ID NO: 4 and thecalcium-insensitive alpha-amylase is SEQ ID NO: 13, and thealpha-amylase optionally further comprises one or more of the followingalterations: D181*+G182*, E187W, E187W+N188E+D191T, N188E, D191T, S299K,S299K+G301R+A302D+D405N+D428N+P430D, G301R, A302D, D405N+D428N, andP430D.Paragraph 7. An isolated alpha-amylase, comprising an A-domain with atleast 60% sequence identity with the A-domain of any of SEQ ID NOS:1-12, 29, and 30, a B-domain with at least 60% sequence identity withthe B-domain of any of SEQ ID NOS: 13-16 and 31, and a C-domain with atleast 60% sequence identity with the C-domain of any of SEQ ID NOS:1-12, 29, and 30.Paragraph 8. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 1.Paragraph 9. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 2.Paragraph 10. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 3.Paragraph 11. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 4.Paragraph 12. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 5.Paragraph 13. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 6.Paragraph 14. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 7.Paragraph 15. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 8.Paragraph 16. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 9.Paragraph 17. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 10.Paragraph 18. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 11.Paragraph 19. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 12.Paragraph 20. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 29.Paragraph 21. The alpha-amylase of paragraph 7, wherein the A-domain hasat least 60% sequence identity, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe A-domain of SEQ ID NO: 30.Paragraph 22. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 91-111,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 96-101 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 101-106, in particular positions 1-101 of SEQ ID NO: 1.Paragraph 23. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 95-115,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 100-105 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 105-110, in particular positions 1-105 of SEQ ID NO: 2.Paragraph 24. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 93-113,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 98-103 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 103-108, in particular positions 1-103 of SEQ ID NO: 3.Paragraph 25. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 94-114,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 99-104 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 104-109, in particular positions 1-104 of SEQ ID NO: 4.Paragraph 26. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 95-115,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 100-105 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 105-110, in particular positions 1-105 of SEQ ID NO: 5.Paragraph 27. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 95-115,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 100-105 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 105-110, in particular positions 1-105 of SEQ ID NO: 6.Paragraph 28. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 95-115,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 100-105 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 105-110, in particular positions 1-105 of SEQ ID NO: 7.Paragraph 29. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 95-115,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 100-105 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 105-110, in particular positions 1-105 of SEQ ID NO: 8.Paragraph 30. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 95-115,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 100-105 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 105-110, in particular positions 1-105 of SEQ ID NO: 9.Paragraph 31. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 95-115,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 100-105 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 105-110, in particular positions 1-105 of SEQ ID NO: 10.Paragraph 32. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 95-115,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 100-105 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 105-110, in particular positions 1-105 of SEQ ID NO: 11.Paragraph 33. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 95-115,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 100-105 or starting at a positionin the range of positions 1-3 and ending at a position in the range ofpositions 105-110, in particular positions 1-105 of SEQ ID NO: 12.Paragraph 34. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 94-114,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 99-104 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 104-109, in particular positions 1-104 of SEQ ID NO: 29.Paragraph 35. The alpha-amylase of any of paragraphs 7-21, wherein theA1-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 1-5 and ending a position in the range of positions 92-112,e.g., starting at a position in the range of positions 1-3 and ending ata position in the range of positions 97-102 or starting at a position inthe range of positions 1-3 and ending at a position in the range ofpositions 102-107, in particular positions 1-102 of SEQ ID NO: 30.Paragraph 36. The alpha-amylase of any of paragraphs 7-35, wherein theB-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 93-113 and ending at a position in the range of positions195-215, e.g., starting at a position in the range of positions 97-109and ending at a position in the range of positions 199-211 or startingat a position in the range of positions 100-106 and ending at a positionin the range of positions 202-208, in particular positions 103-208 ofSEQ ID NO: 13.Paragraph 37. The alpha-amylase of any of paragraphs 7-35, wherein theB-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 93-113 and ending at a position in the range of positions195-215, e.g., starting at a position in the range of positions 97-109and ending at a position in the range of positions 199-211 or startingat a position in the range of positions 100-106 and ending at a positionin the range of positions 202-208, in particular positions 104-207 ofSEQ ID NO: 14.Paragraph 38. The alpha-amylase of any of paragraphs 7-35, wherein theB-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 93-113 and ending at a position in the range of positions195-215, e.g., starting at a position in the range of positions 97-109and ending at a position in the range of positions 199-211 or startingat a position in the range of positions 100-106 and ending at a positionin the range of positions 202-208, in particular positions 104-207 ofSEQ ID NO: 15.Paragraph 39. The alpha-amylase of any of paragraphs 7-35, wherein theB-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 100-120 and ending at a position in the range of positions161-181, e.g., starting at a position in the range of positions 105-115and ending at a position in the range of positions 166-171 or startingat a position in the range of positions 107-113 and ending at a positionin the range of positions 171-176, in particular positions 110-171 ofSEQ ID NO: 16.Paragraph 40. The alpha-amylase of any of paragraphs 7-35, wherein theB-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the sequence starting at a position in the range ofpositions 100-120 and ending at a position in the range of positions161-181, e.g., starting at a position in the range of positions 105-115and ending at a position in the range of positions 166-171 or startingat a position in the range of positions 107-113 and ending at a positionin the range of positions 171-176, in particular positions 110-171 ofSEQ ID NO: 31.Paragraph 41. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 1.Paragraph 42. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 2.Paragraph 43. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 3.Paragraph 44. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 4.Paragraph 45. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 5.Paragraph 46. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 6.Paragraph 47. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 7.Paragraph 48. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 8.Paragraph 49. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 9.Paragraph 50. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 10.Paragraph 51. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 11.Paragraph 52. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 12.Paragraph 53. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 29.Paragraph 54. The alpha-amylase of any of paragraphs 7-40, wherein theC-domain has at least 60% sequence identity, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the C-domain of SEQ ID NO: 30.Paragraph 55. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 198-218 and ending at a position in the range of positions478-483, e.g., starting at a position in the range of positions 203-208and ending at a position in the range of positions 480-483 or startingat a position in the range of positions 208-213 and ending at a positionin the range of positions 480-483, in particular positions 208-483 ofSEQ ID NO: 1.Paragraph 56. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 202-222 and ending at a position in the range of positions479-484, e.g., starting at a position in the range of positions 207-212and ending at a position in the range of positions 481-484 or startingat a position in the range of positions 212-217 and ending at a positionin the range of positions 481-484, in particular positions 212-484 ofSEQ ID NO: 2.Paragraph 57. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 198-218 and ending at a position in the range of positions478-483, e.g., starting at a position in the range of positions 203-208and ending at a position in the range of positions 480-483 or startingat a position in the range of positions 208-213 and ending at a positionin the range of positions 480-483, in particular positions 208-483 ofSEQ ID NO: 3.Paragraph 58. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 201-221 and ending at a position in the range of positions478-483, e.g., starting at a position in the range of positions 206-211and ending at a position in the range of positions 480-483 or startingat a position in the range of positions 211-216 and ending at a positionin the range of positions 480-483, in particular positions 211-483 ofSEQ ID NO: 4.Paragraph 59. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 193-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485, in particular positions 213-485 ofSEQ ID NO: 5.Paragraph 60. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485, in particular positions 213-485 ofSEQ ID NO: 6.Paragraph 61. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485, in particular positions 213-485 ofSEQ ID NO: 7.Paragraph 62. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485, in particular positions 213-485 ofSEQ ID NO: 8.Paragraph 63. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 203-223 and ending at a position in the range of positions481-484, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-484 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-484, in particular positions 213-484 ofSEQ ID NO: 9.Paragraph 64. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 203-223 and ending at a position in the range of positions482-484, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-484 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-484, in particular positions 213-484 ofSEQ ID NO: 10.Paragraph 65. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485, in particular positions 213-485 ofSEQ ID NO: 11.Paragraph 66. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 203-223 and ending at a position in the range of positions482-485, e.g., starting at a position in the range of positions 208-213and ending at a position in the range of positions 482-485 or startingat a position in the range of positions 213-218 and ending at a positionin the range of positions 482-485, in particular positions 213-485 ofSEQ ID NO: 12.Paragraph 67. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 201-221 and ending at a position in the range of positions478-483, e.g., starting at a position in the range of positions 206-211and ending at a position in the range of positions 480-483 or startingat a position in the range of positions 211-216 and ending at a positionin the range of positions 480-483, in particular positions 211-483 ofSEQ ID NO: 29.Paragraph 68. The alpha-amylase of any of paragraphs 7-54, wherein theA2 and C-domains have at least 60% sequence identity, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the sequence starting at a position in the rangeof positions 199-219 and ending at a position in the range of positions479-484, e.g., starting at a position in the range of positions 204-209and ending at a position in the range of positions 481-484 or startingat a position in the range of positions 209-214 and ending at a positionin the range of positions 481-484, in particular positions 209-484 ofSEQ ID NO: 30.Paragraph 69. The alpha-amylase of any of paragraphs 1-68, which is morethermostable than the alpha-amylase of any of SEQ ID NOS: 1-12, 29 and30.Paragraph 70. The alpha-amylase of any of paragraphs 1-69, which hasreduced calcium sensitivity than the alpha-amylase of any of SEQ ID NOS:1-12, 29 and 30.Paragraph 71. A detergent composition comprising an alpha-amylase of anyof paragraphs 1-70 and a surfactant.Paragraph 72. A composition comprising an alpha-amylase of any ofparagraphs 1-70 and one or more enzymes selected from the groupconsisting of beta-amylase, cellulase (beta-glucosidase,cellobiohydrolase, and endoglucanase) glucoamylase, hemicellulase (e.g.,xylanase), isoamylase, isomerase, lipase, phytase, protease, andpullulanase.Paragraph 73. Use of an alpha-amylase of any of paragraphs 1-70 forwashing and/or dishwashing.Paragraph 74. Use of an alpha-amylase of any of paragraphs 1-70 fordesizing a textile.Paragraph 75. Paragraph 89. Use of an alpha-amylase of any of paragraphs1-70 for producing a baked product.Paragraph 76. Use of an alpha-amylase of any of paragraphs 1-70 forliquefying a starch-containing material.Paragraph 77. A method of producing liquefied starch, comprisingliquefying a starch-containing material with an alpha-amylase of any ofparagraphs 1-70.Paragraph 78. A process of producing a fermentation product, comprising

(a) liquefying a starch-containing material with an alpha-amylase of anyof paragraphs 1-70 to produce a liquefied mash;

(b) saccharifying the liquefied mash to produce fermentable sugars; and

(c) fermenting the fermentable sugars in the presence of a fermentingorganism.

Paragraph 79. The process of paragraph 78, wherein the starch-containingmaterial is liquefied with the alpha-amylase and a pullulanase, e.g., aGH57 pullulanase.

Paragraph 80. The process of paragraph 79, wherein the pullulanase isobtained from a strain of Thermococcus, including Thermococcus sp. AM4,Thermococcus sp. HJ21, Thermococcus barophilus, Thermococcusgammatolerans, Thermococcus hydrothermalis; Thermococcus kodakarensis,Thermococcus litoralis, and Thermococcus onnurineus; or from a strain ofPyrococcus, such as Pyrococcus abyssi and Pyrococcus furiosus.Paragraph 81. The process of any of paragraphs 78-80, further comprisingadding a protease, such as an acid fungal protease or a metalloproteasebefore, during and/or after liquefaction.Paragraph 82. The process of paragraph 81, wherein the metalloproteaseis obtained from a strain of Thermoascus, preferably a strain ofThermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No.0670.Paragraph 83. A process of producing a fermentation product, comprisingcontacting a starch substrate with an alpha-amylase of any of paragraphs1-70, a glucoamylase, and a fermenting organism.Paragraph 84. The process of any of paragraphs 78-83, wherein thefermentation product is selected from the group consisting of alcohol(e.g., ethanol and butanol), organic acids (e.g., succinic acid andlactic acid), sugar alcohols (e.g., glycerol), ascorbic acidintermediates (e.g., gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g.,lysine), proteins (e.g., antibodies and fragment thereof).Paragraph 85. A nucleic acid sequence encoding an alpha-amylase of anyof paragraphs 1-70.Paragraph 86. A plasmid comprising the nucleic acid sequence ofparagraph 85.Paragraph 87. A host cell comprising the nucleic acid sequence ofparagraph 85 or a plasmid of paragraph 86.Paragraph 88. A transgenic plant, plant part or plant cell transformedwith the nucleic acid sequence of paragraph 85.Paragraph 89. A method for preparing an alpha-amylase of any ofparagraphs 1-70, comprising the following steps:

(a) growing the host cell of paragraph 87 under conditions leading toexpression of the hybrid alpha-amylase; and

(b) recovering the hybrid alpha-amylase.

The invention claimed is:
 1. An isolated alpha-amylase comprising the A-and C-domains of a calcium-sensitive alpha-amylase and the B-domain of acalcium-insensitive alpha-amylase, wherein the A- and C-domains have atleast 90% sequence identity with the A- and C-domains of SEQ ID NO: 5,and the B-domain has at least 90% sequence identity with the B-domain ofSEQ ID NO:
 13. 2. An isolated alpha-amylase, comprising an A-domain withat least 90% sequence identity with the A-domain of SEQ ID NO: 5, aB-domain with at least 90% sequence identity with the B-domain of SEQ IDNO: 13, and a C-domain with at least 90% sequence identity with theC-domain of SEQ ID NOS:
 5. 3. The alpha-amylase of claim 2, wherein theA-domain has at least 95 sequence identity with the A-domain of SEQ IDNO:
 5. 4. The alpha-amylase of claim 1, wherein the A-domain has atleast 95% sequence identity with the sequence starting at a position inthe range of positions 1-5 and ending a position in the range ofpositions 94-114 of SEQ ID NO:
 5. 5. The alpha-amylase of claim 1,wherein the B-domain has at least 95% sequence identity with thesequence starting at a position in the range of positions 93-113 andending at a position in the range of positions 195-215 of SEQ ID NO: 13.6. The alpha-amylase of claim 1, wherein the C-domain has at least 95%sequence identity with the C-domain of SEQ ID NO:
 5. 7. Thealpha-amylase of claim 1, which is more thermostable than thealpha-amylase of any of SEQ ID NOS: 1-12, 29 and
 30. 8. Thealpha-amylase of claim 1, which has reduced calcium sensitivity than thealpha-amylase of any of SEQ ID NOS: 1-12, 29 and
 30. 9. A detergentcomposition comprising an alpha-amylase of claim 1 and a surfactant. 10.A composition comprising an alpha-amylase of claim 1 and one or moreenzymes selected from the group consisting of beta-amylase, cellulaseglucoamylase, hemicellulase, isoamylase, isomerase, lipase, phytase,protease, and pullulanase.
 11. The alpha-amylase of claim 2, wherein theB-domain has at least 95% sequence identity with the B-domain of SEQ IDNO:
 13. 12. The alpha-amylase of claim 2, wherein the C-domain has atleast 95% sequence identity with the C-domain of SEQ ID NO:
 5. 13. Thecomposition of claim 10, wherein the cellulase is selected from thegroup consisting of beta-glucosidase, cellobiohydrolase, andendoglucanase.
 14. The composition of claim 10, wherein thehemicellulase is a xylanase.